Bacterial strains with toxin complex for insect control

ABSTRACT

Biological strains, compositions, and methods of using the strains and compositions for reducing overall insect damage.

FIELD

Biological strains, compositions, and methods of using the strains andcompositions for reducing overall insect damage. Also provided are novelgenes that encode pesticidal proteins. These pesticidal proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named8472_SeqList.txt created on Oct. 8, 2020 and having a size of 344kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

BACKGROUND

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains of Bt.These genetically modified crops are now widely used in agriculture andhave provided the farmer with an environmentally friendly alternative totraditional insect-control methods. While they have proven to be verysuccessful commercially, these genetically modified, insect-resistantcrop plants provide resistance to only a narrow range of theeconomically important insect pests. In some cases, insects can developresistance to different insecticidal compounds, which raises the need toidentify alternative biological control agents for pest control.

There has been a long felt need for environmentally friendlycompositions and methods for controlling or eradicating insect pests ofagricultural significance, i.e., methods that are selective,environmentally inert, non-persistent, and biodegradable, and that fitwell into insect pest management schemes.

SUMMARY

Some embodiments relate to a composition comprising a Pantoeaagglomerans, wherein the Pantoea agglomerans has insecticidal activity.In some embodiments, the methods and compositions relate to ainsecticidal bacterial strain comprising IPD126 gene or gene cluster. Insome embodiments, the IPD126 gene comprises an amino acid sequencehaving at least 90% sequence identity to any one of SEQ ID NOs: 19-36.In some embodiments, the methods and compositions relate to bacterialstrains comprising a 16S RNA sequence having at least 95% identity toany one of SEQ ID NOs: 37-39.

In one embodiment, the disclosure relates to a composition comprising aPantoea agglomerans strain PMC3671E3-1 (NRRL Deposit No. B-67697),wherein the Pantoea agglomerans strain PMC3671E3-1 has insecticidalactivity.

In one embodiment, the disclosure relates to a composition comprising aPantoea agglomerans strain PMC3671E9-1 (NRRL Deposit No. B-67698),wherein the Pantoea agglomerans strain PMC3671E9-1 has insecticidalactivity.

In one embodiment, the disclosure relates to a composition comprising aPantoea agglomerans strain PMCJ4082D4-1 (NRRL Deposit No. B-67699),wherein the Pantoea agglomerans strain PMCJ4082D4-1 has insecticidalactivity.

In yet another embodiment, the disclosure relates methods andcompositions comprising a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof in an effectiveamount to achieve an effect of inhibit growth of a plant pathogen, pestor insect. In another embodiment, the composition further comprises abiocontrol agent selected from the group consisting of bacteria, fungi,yeast, protozoans, viruses, entomopathogenic nematodes, botanicalextracts, proteins, secondary metabolites, and inoculants.

In another embodiment, the compositions and methods disclosed hereinfurther comprise one or more agrochemically active compounds selectedfrom the group consisting of an insecticide, a fungicide, a bactericide,and a nematicide. In still another embodiment, the composition furthercomprises a compound selected from the group consisting of a safener, alipo-chitooligosaccharide, an isoflavone, and a ryanodine receptormodulator.

In another embodiment, the compositions and methods comprise at leastone at least one seed, plant, or plant part. In one embodiment, theseed, plant, or plant part is genetically modified.

In one embodiment, the compositions and methods inhibit the growth ofone or more plant pathogens, pests, or insects including but not limitedto bacteria, a fungus, a virus, protozoa, nematode, or an arthropod. Inone embodiment, the compositions and methods inhibit the growth of aninsect, including but not limited to a Coleopteran, Hemipteran, orLepidopteran insect. In still another embodiment, the compositioninhibits the growth of Diabrotica virgifera virgifera, Ostrinianubilalis, Spodoptera frugiperda, Pseudoplusia includens, Anticarsiagemmatalis, Plutella xylostella, and/or Aphis fabae.

In another embodiment, the compositions and methods comprise aneffective amount to provide pesticidal activity to bacteria, plants,plant cells, tissues and seeds. In another embodiment, the compositionis an effective amount to provide pesticidal activity to Coleopteran orLepidopteran insects. In still another embodiment, the composition is aneffective amount to provide pesticidal activity to Diabrotica virgiferavirgifera, Ostrinia nubilalis, Spodoptera frugiperda, Pseudoplusiaincludens, Anticarsia gemmatalis, Plutella xylostella, and/or Aphisfabae.

In another embodiment, the compositions and methods comprise in aneffective amount to improve plant performance including but not limitedto increased root formation, increased root mass, increased rootfunction, increased shoot height, increased shoot function, increasedflower bud presence, increased flower bud formation, increased seedgermination, increased yield, increased total plant wet weight, andincreased total plant dry weight.

In another embodiment, the disclosure relates to a method comprisingapplying a composition comprising a bacterial strain disclosed herein,or a progeny, mutant, or variant thereof, a fermentate produced from astrain disclosed herein, or a progeny, mutant, or variant thereof, aplant, plant part or soil in an effective amount to achieve an effectselected from the group consisting of: inhibit a plant pathogen, pest,or insect or to prevent damage to a plant by a pathogen, pest, orinsect; improve plant performance; improve plant yield; improve plantvigor; increase phosphate availability; increase production of a planthormone; increase root formation; increase shoot height in a plant,increase leaf length of a plant; increase flower bud formation of aplant; increase total plant fresh weight; increase total plant dryweight; and increase seed germination.

In another embodiment, the method further comprises applying abiocontrol agent, wherein the biocontrol agent is selected from thegroup consisting of bacteria, fungi, yeast, protozoans, viruses,entomopathogenic nematodes, botanical extracts, proteins, secondarymetabolites, and inoculants.

In yet another embodiment, the method further comprises applying anagrochemically active compound selected from the group consisting of aninsecticide, a fungicide, a bactericide, and a nematicide.

In still another embodiment, the method further comprises applying acompound selected from the group consisting of a safener, alipo-chitooligosaccharide, an isoflavone, and a ryanodine receptormodulator.

In another embodiment, the method comprises applying the composition inan effective amount to inhibit growth of a plant pathogen, including butnot limited to bacteria, a fungus, a nematode, an insect, a virus, andprotozoa.

In one aspect compositions and methods for conferring pesticidalactivity to bacteria, plants, plant cells, tissues and seeds areprovided. Compositions include nucleic acid molecules encoding sequencesfor pesticidal and insecticidal polypeptides, vectors comprising thosenucleic acid molecules, and host cells comprising the vectors.Compositions also include the pesticidal polypeptide sequences andantibodies to those polypeptides. Compositions also comprise transformedbacteria, plants, plant cells, tissues and seeds.

In another aspect methods are provided for producing the polypeptidesand for using those polypeptides for controlling or killing aHemipteran, Coleopteran, Lepidopteran, or nematode pests. The transgenicplants of the embodiments express one or more of the pesticidalsequences disclosed herein. In various embodiments, the transgenic plantfurther comprises one or more additional genes for insect resistance,for example, one or more additional genes for controlling Hemipteran,Coleopteran, Lepidopteran, or nematode pests. It will be understood byone of skill in the art that the transgenic plant may comprise any geneimparting an agronomic trait of interest.

In another aspect methods for detecting the nucleic acids andpolypeptides of the embodiments in a sample are also included. A kit fordetecting the presence of an IPD126 polypeptide or detecting thepresence of a polynucleotide encoding an IPD126 polypeptide in a sampleis provided. The kit may be provided along with all reagents and controlsamples necessary for carrying out a method for detecting the intendedagent, as well as instructions for use.

In another aspect, the compositions and methods of the embodiments areuseful for the production of organisms with enhanced pest resistance ortolerance. These organisms and compositions comprising the organisms aredesirable for agricultural purposes. The compositions of the embodimentsare also useful for generating altered or improved proteins that havepesticidal activity or for detecting the presence of IPD126polypeptides.

DESCRIPTION OF FIGURES

FIG. 1 shows IPD126 gene clusters in insecticidal strains of Pantoeaagglomerans.

DESCRIPTION OF THE SEQUENCES

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing that forma part of this application.

The sequence descriptions summarize the Sequence Listing attachedhereto, which is hereby incorporated by reference. The Sequence Listingcontains one letter codes for nucleotide sequence characters and thesingle and three letter codes for amino acids as defined in theIUPAC-IUB standards described in Nucleic Acids Research 13:3021-3030(1985) and in the Biochemical Journal 219(2):345-373 (1984).

TABLE 1 Sequence Listing Description Strain_Gene SEQ ID NO:PMC3671E9-1_IPD126Aa-1 DNA  1 PMC3671E9-1_IPD126Aa-2 DNA  2PMC3671E9-1_IPD126Aa-3 DNA  3 PMC3671E9-1_IPD126Aa-4 DNA  4PCM3671E3-1_IPD126Aa-1 DNA  5 PCM3671E3-1_IPD126Aa-2 DNA  6PCM3671E3-1_IPD126Aa-3 DNA  7 PCM3671E3-1_IPD126Aa-4 DNA  8PCM3671E3-1_IPD126Aa-1.2 DNA  9 PCM3671E3-1_IPD126Aa-2.2 DNA 10PCM3671E3-1_IPD126Aa-3.2 DNA 11 PMCJ4082D4-1_IPD126Aa-1 DNA 12PMCJ4082D4-1_IPD126Aa-2 DNA 13 PMCJ4082D4-1_IPD126Aa-3 DNA 14PMCJ4082D4-1_IPD126Aa-4 DNA 15 PMCJ4082D4-1_IPD126Aa-1.2 DNA 16PMCJ4082D4-1_IPD126Aa-2.2 DNA 17 PMCJ4082D4-1_IPD126Aa-3.2 DNA 18PMC3671E9-1_IPD126Aa-1 protein 19 PMC3671E9-1_IPD126Aa-2 Protein 20PMC3671E9-1_IPD126Aa-3 Protein 21 PMC3671E9-1_IPD126Aa-4 Protein 22PCM3671E3-1_IPD126Aa-1 protein 23 PCM3671E3-1_IPD126Aa-2 protein 24PCM3671E3-1_IPD126Aa-3 Protein 25 PCM3671E3-1_IPD126Aa-4 Protein 26PCM3671E3-1_IPD126Aa-1.2 protein 27 PCM3671E3-1_IPD126Aa-2.2 Protein 28PCM3671E3-1_IPD126Aa-3.2 Protein 29 PMCJ4082D4-1_IPD126Aa-1 Protein 30PMCJ4082D4-1_IPD126Aa-2 protein 31 PMCJ4082D4-1_IPD126Aa-3 protein 32PMCJ4082D4-1_IPD126Aa-4 protein 33 PMCJ4082D4-1_IPD126Aa-1.2 protein 34PMCJ4082D4-1_IPD126Aa-2.2 protein 35 PMCJ4082D4-1_IPD126Aa-3.2 protein36 PMC3671E3-1-16S 37 PMC3671E9-1-16S 38 PMCJ4082D4-1-16S 39

DETAILED DESCRIPTION

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof, and so forth. All technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this disclosure belongs unlessclearly indicated otherwise.

As used herein, “administer” refers to the action of introducing astrain and/or a composition to an environment for pathogen, pest, orinsect inhibition or to improve plant performance.

As used herein, the term “agrochemically active compounds” refers to anysubstance that is or may be customarily used for treating plantsincluding, but not limited to, fungicides, bactericides, insecticides,acaricides, nematicides, molluscicides, safeners, plant growthregulators, and plant nutrients, as well as, microorganisms.

As used herein, a composition may be a liquid, a heterogeneous mixture,a homogeneous mixture, a powder, a solution, a dispersion or anycombination thereof.

As used herein, “effective amount” refers to a quantity of a bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof sufficient to inhibit growth of a pathogenicmicroorganism or to impede the rate of growth of the pathogenicmicroorganism. In another embodiment, the term “effective amount” refersto a quantity of a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof sufficient toimprove plant performance. In another embodiment, the term “effectiveamount” refers to a quantity of a bacterial strain disclosed herein, ora progeny, mutant, or variant thereof, a fermentate produced from astrain disclosed herein progeny, mutant, or variant thereof, sufficientto control, kill, inhibit, and reduce the number, emergence, or growthof a pathogen, pest, or insect. In another embodiment, the term“effective amount” refers to a quantity of a bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, a fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereofsufficient to prevent damage from a pathogen, pest, or insect. Oneskilled in the art will recognized that an effective amount of abacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof may not reduce the numbers of pathogens,pests or insects, but is effective in decreasing damage to plants and/orplant parts as a result of a pathogen, pest or insect. For example, apesticidally effective amount may reduce pathogen, pest or insectemergence, or damage to seeds, roots, shoots, or foliage of plants thatare treated compared to those that are untreated.

As used herein, “fermentate broth,” “fermentate,” or “fermented broth”refers to a media. used to grow or ferment a bacterial strain disclosedherein. The bacterial strain may be removed from a media by filtration,sterilization, or other means. The leftover broth contains metabolitesproduced by a bacterial strain disclosed herein, which is collectivelyreferred to as a “fermentate broth,” “fermentate,” or “fermented broth.”

As used herein, the term “inhibit” refers to destroy, prevent, reduce,resist, control, decrease, slow or otherwise interfere with the growthor survival of a pathogen, pest, or insect when compared to the growthor survival of the pathogen, pest, or insect in an untreated control.Any of the terms of inhibit, destroy, prevent, control, decrease, slow,interfere, resist, or reduce may be used interchangeably. In oneembodiment, to “inhibit” is to destroy, prevent, control, reduce,resist, decrease, slow or otherwise interfere with the growth,emergence, or survival of a pathogen, pest, or insect by at least about3% to at least about 100%, or any value in between for example at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth orsurvival of the pathogen, pest, or insect in an untreated control. Theamount of inhibition can be measured as described herein or by othermethods known in the art. As used herein, “protects a plant from apathogen, pest, or insect pest” is intended to mean the limiting oreliminating of the pathogen, pest, or insect related damage to a plantand/or plant part by, for example, inhibiting the ability of thepathogen, pest, or insect to grow, emerge, feed, and/or reproduce or bykilling the pathogen, pest, or insect. As used herein, pesticidal and/orinsecticidal activity refers to an activity of compound, composition,and or method that protects a plant and/or plant part from a pathogen,pest, or insect.

In some embodiments, inhibition a pathogen, pest, or insect lasts for orprovides protection for greater than a day, two days, three days, fourdays, five days, six days, a week, two weeks, three weeks, a month ormore after of a bacterial strain disclosed herein, or a progeny, mutant,or variant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof disclosed herein is applied tosubject material. In another embodiment, inhibition a pathogen, pest orinsect lasts from one to seven days, from seven to 14 days, from 14 to21 days, or from 21 to 30 days or more. In another embodiment, theinhibition of the growth of a pathogen, pest, or insect lasts for orprovides protection for greater than the time from application to adultemergence of the pathogen, pest, or insect.

As used herein, the term “genetically modified” is intended to mean anyspecies containing a genetic trait, loci, or sequence that was not foundin the species or strain prior to manipulation. A genetically modifiedplant may be transgenic, cis-genic, genome edited, or bred to contain anew genetic trait, loci, or sequence. A genetically modified plant orbacteria may be prepared by means known to those skilled in the art,such as transformation by bombardment, by a gene editing technique suchas Cas/CRISPR or TALENS, or by breeding techniques. As used herein, a“trait” is a new or modified locus or sequence of a genetically modifiedplant or bacteria, including but not limited to a transgenic plant orbacteria. In some embodiments, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, may be edited. In some embodimentsany bacterial strain may be modified or edited to comprise an IPD126gene. In some embodiments, the methods and compositions relate to aninsecticidal bacterial strain comprising an IPD126 gene

As used herein, the term “environment of a plant or plant part” isintended to mean the area surrounding the plant or plant part, includingbut not limited to the soil, the air, or in-furrow. The environment of aplant or plant part may be in proximity, touching, adjacent to, or inthe same field as the plant or plant part. The compositions describedherein may be applied to the environment of the plant or plant part as aseed treatment, as a foliar application, as a granular application, as asoil application, or as an encapsulated application. As used herein,“in-furrow” is intended to mean within or near the area where a seed isplanted. The compositions disclosed herein may be applied in-furrowconcurrently or simultaneously with a seed. In another embodiment, thecompositions disclosed herein may be applied sequentially, either beforeor after a seed is planted.

As used herein, the term “different mode of action” is used to refer toa pesticidal composition inhibiting a pathogen, pest, or insect througha pathway or receptor that is different from another pesticidalcomposition. As used herein, the term “different mode of action”includes the pesticidal effects of one or more pesticidal compositionsto different binding sites (i.e., different toxin receptors and/ordifferent sites on the same toxin receptor) in the gut membranes ofinsects or through the RNA interference pathway to different targetgenes.

As used herein, the term “pathogen, pest, or insect” includes but is notlimited to pathogenic fungi, bacteria, mites, ticks, pathogenicmicroorganisms, and nematodes, as well as insect from the ordersColeoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonatpera, Trichoptera,and others, including but not limited to Diabrotica virgifera virgifera,Diabrotica undecimpunctata howardi, and Diabrotica barberi.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermüller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Géhin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Melanaphis sacchari (sugarcane aphid); Adelges spp. (adelgids);Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaciGennadius (tobacco whitefly, sweetpotato whitefly); B. argentifoliiBellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead(citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) andT. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris(potato leafhopper); Laodelphax striatellus Fallen (smaller brownplanthopper); Macrolestes quadrilineatus Forbes (aster leafhopper);Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal(rice leafhopper); Nilaparvata lugens Stal (brown planthopper);Peregrinus maidis Ashmead (corn planthopper); Sogatella furciferaHorvath (white-backed planthopper); Sogatodes orizicola Muir (ricedelphacid); Typhlocyba pomaria McAtee (white apple leafhopper);Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus(periodical cicada); Icerya purchasi Maskell (cottony cushion scale);Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citriRisso (citrus mealybug); Pseudococcus spp. (other mealybug complex);Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead(persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schïffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477. Generally, the pesticide is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

“Percent (%) sequence identity” with respect to a reference sequence(subject) is determined as the percentage of amino acid residues ornucleotides in a candidate sequence (query) that are identical with therespective amino acid residues or nucleotides in the reference sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyamino acid conservative substitutions as part of the sequence identity.Alignment for purposes of determining percent sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (e.g., percentidentity of query sequence=number of identical positions between queryand subject sequences/total number of positions of query sequence×100).

In some embodiments, an IPD126 polypeptide comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greateridentity across the entire length of the amino acid sequence of any oneof SEQ ID NOs: 19-36. In some embodiments, a nucleic acid sequenceencoding an IPD126 polynucleotide sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater identity across the entire length ofthe amino acid sequence of any one of SEQ ID NOs: 1-18.

As used herein, the term “plant” refers to all plants, plant parts,seed, and plant populations, such as desirable and undesirable wildplants, cultivars, transgenic plants, and plant varieties (whether ornot protectable by plant variety or plant breeder's rights). Cultivarsand plant varieties can be plants obtained by conventional propagationand breeding methods that can be assisted or supplemented by one or morebiotechnological methods such as by use of double haploids, protoplastfusion, random and directed mutagenesis, molecular or genetic markers orby bioengineering and genetic engineering methods.

The embodiments disclosed herein may generally be used for any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables ornamentals, and conifers.

As used herein, the term “plant parts” refers to all above ground andbelow ground parts and organs of plants such as shoot, leaf, blossom androot, whereby for example leaves, needles, stems, branches, blossoms,fruiting bodies, fruits and seeds, as well as roots, tubers, corms andrhizomes are included. Crops and vegetative and generative propagatingmaterial, for example, cuttings, corms, rhizomes, tubers, runners andseeds are also plant parts.

As used herein, the term “viable” refers to a microbial cell, propagule,or spore that is metabolically active or able to differentiate. Thus,propagules, such as spores, are “viable” when they are dormant andcapable of germinating.

The embodiments disclosed herein relate to a Pantoea agglomerans strainPMC3671E3-1 (NRRL Deposit No. B-67697), a Pantoea agglomerans strainPMC3671E9-1 (NRRL Deposit No. B-67698), or a Pantoea agglomerans strainPMCJ4082D4-1 (NRRL Deposit No. B-67699); and/or a fermentate producedfrom a growth medium comprising a Pantoea agglomerans strain PMC3671E3-1(NRRL Deposit No. B-67697), a Pantoea agglomerans strain PMC3671E9-1(NRRL Deposit No. B-67698), or a Pantoea agglomerans strain PMCJ4082D4-1(NRRL Deposit No. B-67699. In one embodiment the bacterial straindisclosed herein, or a progeny, mutant, or variant thereof; and/or afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof; compositions and methods find use in inhibiting,controlling, or killing a pathogen, pest, or insect, including, but isnot limited to, fungi, pathogenic fungi, bacteria, mites, ticks,pathogenic microorganisms, and nematodes, as well as insects from theorders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera, Isoptera,Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera,including but not limited to Diabrotica virgifera virgifera, Diabroticaundecimpunctata howardi, and Diabrotica barberi, and for producingcompositions with pesticidal activity.

The Pantoea agglomerans strain PMC3671E3-1 (NRRL Deposit No. B-67697),Pantoea agglomerans strain PMC3671E9-1 (NRRL Deposit No. B-67698), andPantoea agglomerans strain PMCJ4082D4-1 (NRRL Deposit No. B-67699) weredeposited on Nov. 9, 2018 at the Agricultural Research Service CultureCollection (NRRL), 1815 North University Street, Peoria, Ill., 61604.The deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. Further, these deposits will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.Access to these deposits will be available during the pendency of theapplication to the Commissioner of Patents and Trademarks and personsdetermined by the Commissioner to be entitled thereto upon request. Uponallowance of any claims in the application, the Applicant will makeavailable to the public, pursuant to 37 C.F.R. § 1.808, sample(s) of thedeposits. The deposits will be maintained in the NRRL depository, whichis a public depository, for a period of 30 years, or 5 years after themost recent request, or for the enforceable life of the patent,whichever is longer, and will be replaced if it becomes nonviable duringthat period. Additionally, Applicant has satisfied all the requirementsof 37 C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample upon deposit.

Some embodiments relate to compositions comprising or consisting of orconsisting essentially of a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof. In one embodiment,the compositions are biologically pure cultures of the strain disclosedherein.

Some embodiments relate to a composition comprising a bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof disclosed herein and one or more compounds or agents selectedfrom the group consisting of: agrochemically active compounds,biocontrol agents, lipo-chitooligosaccharide compounds (LCOs),isoflavones, quinazolines, insecticidal compounds,azolopyrimidinylamines, polymeric compounds, ionic compound, substitutedthiophenes, substituted dithiines, fluopyramm, enaminocarbonylcompounds, strigolactone compound, and dithiino-tetracarboximidecompounds.

A further embodiment relates to the use of a first compositioncomprising a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof disclosed herein and a secondcomposition comprising one or more compounds or agents selected from thegroup consisting of: agrochemically active compounds, biocontrol agents,lipo-chitooligosaccharide compounds (LCOs), isoflavones, quinazolines,insecticidal compound, azolopyrimidinylamine, polymeric compounds, ioniccompound, substituted thiophenes, substituted dithiines, fluopyramm,enaminocarbonyl compounds, strigolactone compound, anddithiino-tetracarboximide compounds.

In one embodiment, the disclosure relates to a composition comprising abacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof disclosed herein and one or more biocontrolagents. As used herein, the term “biocontrol agent” (“BCA”) includesbacteria, fungi or yeasts, protozoans, viruses, entomopathogenicnematodes, and botanical extracts, or products produced bymicroorganisms including proteins or secondary metabolite, andinoculants that have one or both of the following characteristics: (1)inhibits or reduces plant infestation and/or growth of pathogens, pests,or insects, including but not limited to pathogenic fungi, bacteria, andnematodes, as well as arthropod pests such as insects, arachnids,chilopods, diplopods, or that inhibits plant infestation and/or growthof a combination of plant pathogens, pests, or insects; (2) improvesplant performance; (3) improves plant yield; (4) improves plant vigor;and (5) improves plant health.

In one embodiment, the disclosure relates to a composition comprising abacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof disclosed herein and one or moreagrochemically active compounds. Agrochemically active compounds aresubstances that are or may be used for treating a seed, a plant, plantpart, or the environment of the seed or plant or plant part includingbut not limited to fungicides, bactericides, insecticides, acaricides,nematicides, molluscicides, safeners, plant growth regulators, plantnutrients, chemical entities with a known mechanism of action,additional microorganisms, and biocontrol agents.

In another embodiment, the disclosure relates to a first compositioncomprising a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof and a second composition comprisingone or more agrochemically active compounds, wherein the first andsecond composition may inhibit plant pathogens, pests, or insects and/orimprove plant performance.

In one embodiment, the first and second compositions can be applied atthe same time to a seed, a plant, plant part, or the environment of theplant. In another embodiment, the first composition can be applied tothe seed followed by application of the second composition to the seed.In yet another embodiment, the second composition can be applied to theseed followed by, application of the first composition to the seed.

In another embodiment, the first composition can be applied to the plantor plant part followed by application of the second composition to theplant or plant part. In yet another embodiment, the second compositioncan be applied to the plant or plant part followed by application of thefirst composition to the plant or plant part.

In another embodiment, the first composition can be applied to the seedand the second composition applied to the plant or plant part. In yetanother embodiment, the second composition can be applied to the seedand the first composition applied to the plant or plant part.

In another embodiment, the first composition may be planted on or nearthe seed in a field. In yet another embodiment, the second compositioncan be applied to the seed and the first composition applied to theplant or plant part.

In one embodiment, the disclosure relates to the use of a bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein, progeny, mutant, orvariant thereof, disclosed herein with a composition comprising aninsecticidal protein from Pseudomonas sp. such as PSEEN3174 (Monalysin;(2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHA0and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) EnvironmentalMicrobiology 10:2368-2386; GenBank Accession No. EU400157); fromPseudomonas Taiwanensis (Liu, et al., (2010) J. Agric. Food Chem.,58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al.,(2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant CellTiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdussp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open ToxicologyJournal, 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1polypeptide of U.S. Pat. No. 9,688,730; an AfIP-1A and/or AfIP-1Bpolypeptide of U.S. Pat. No. 9,475,847; a PIP-47 polypeptide of USPublication Number US20160186204; an IPD045 polypeptide, an IPD064polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077polypeptide of PCT Publication Number WO 2016/114973; an IPD080polypeptide of PCT Serial Number PCT/US17/56517; an IPD078 polypeptide,an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, anIPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide ofSerial Number PCT/US17/54160; PIP-72 polypeptide of US PatentPublication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65polypeptide of US Publication Number US20170166921; an IPD098polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109polypeptide of U.S. Ser. No. 62/521,084; a PtIP-83 polypeptide of USPublication Number US20160347799; a PtIP-96 polypeptide of USPublication Number US20170233440; an IPD079 polypeptide of PCTPublication Number WO2017/23486; an IPD082 polypeptide of PCTPublication Number WO 2017/105987, an IPD090 polypeptide of SerialNumber PCT/US17/30602, an IPD093 polypeptide of U.S. Ser. No.62/434,020; an IPD103 polypeptide of Serial Number PCT/US17/39376; anIPD101 polypeptide of U.S. Ser. No. 62/438,179; an IPD121 polypeptide ofUS Serial Number U.S. 62/508,514; and δ-endotoxins including, but notlimited to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9,Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19,Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29,Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39,Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry51 andCry55 classes of δ-endotoxin genes and the B. thuringiensis cytolyticCyt1 and Cyt2 genes. Other Cry proteins are well known to one skilled inthe art (see, Crickmore, et al., “Bacillus thuringiensis toxinnomenclature” (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/which can be accessed on the world-wide web using the “www” prefix). Theinsecticidal activity of Cry proteins is well known to one skilled inthe art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path.101:1-16).

In one embodiment the composition comprises a silencing element of oneor more polynucleotides of interest resulting in suppression of one ormore target pathogen, pest, or insect polypeptides. By “silencingelement” is it intended to mean a polynucleotide which when contacted byor ingested by a pest, is capable of reducing or eliminating the levelor expression of a target polynucleotide or the polypeptide encodedthereby. The silencing element employed can reduce or eliminate theexpression level of the target sequence by influencing the level of thetarget RNA transcript or, alternatively, by influencing translation andthereby affecting the level of the encoded polypeptide. Silencingelements may include, but are not limited to, a sense suppressionelement, an antisense suppression element, a double stranded RNA, asiRNA, an amiRNA, a miRNA, or a hairpin suppression element.

Nucleic acid molecules including silencing elements for targeting thevacuolar ATPase H subunit, useful for controlling a coleopteran pestpopulation and infestation as described in US Patent ApplicationPublication 2012/0198586. PCT Publication WO 2012/055982 describesribonucleic acid (RNA or double stranded RNA) that inhibits or downregulates the expression of a target gene that encodes: an insectribosomal protein such as the ribosomal protein L19, the ribosomalprotein L40 or the ribosomal protein S27A; an insect proteasome subunitsuch as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasomebeta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomerof the COPI vesicle, the γ-coatomer of the COPI vesicle, the β′-coatomerprotein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2A protein which is a putative transmembrane domain protein; an insectprotein belonging to the actin family such as Actin 5C; an insectubiquitin-5E protein; an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport; an insectcrinkled protein which is an unconventional myosin which is involved inmotor activity; an insect crooked neck protein which is involved in theregulation of nuclear alternative mRNA splicing; an insect vacuolarH+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-bindingprotein. PCT publication WO 2007/035650 describes ribonucleic acid (RNAor double stranded RNA) that inhibits or down regulates the expressionof a target gene that encodes Snf7. US Patent Application publication2011/0054007 describes polynucleotide silencing elements targetingRPS10. US Patent Application publication 2014/0275208 describespolynucleotide silencing elements targeting RyanR and PAT3. US PatentApplication Publications 2012/029750, US 20120297501, and 2012/0322660describe interfering ribonucleic acids (RNA or double stranded RNA) thatfunctions upon uptake by an insect pest species to down-regulateexpression of a target gene in said insect pest, wherein the RNAcomprises at least one silencing element wherein the silencing elementis a region of double-stranded RNA comprising annealed complementarystrands, one strand of which comprises or consists of a sequence ofnucleotides which is at least partially complementary to a targetnucleotide sequence within the target gene. US Patent ApplicationPublication 2012/0164205 describe potential targets for interferingdouble stranded ribonucleic acids for inhibiting invertebrate pestsincluding: a Chd3 Homologous Sequence, a Beta-Tubulin HomologousSequence, a 40 kDa V-ATPase Homologous Sequence, a EF1α HomologousSequence, a 26S Proteosome Subunit p28 Homologous Sequence, a JuvenileHormone Epoxide Hydrolase Homologous Sequence, a Swelling DependentChloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate1-Dehydrogenase Protein Homologous Sequence, an Act42A ProteinHomologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, aTranscription Factor IIB Protein Homologous Sequence, a ChitinaseHomologous Sequences, a Ubiquitin Conjugating Enzyme HomologousSequence, a Glyceraldehyde-3-Phosphate Dehydrogenase HomologousSequence, an Ubiquitin B Homologous Sequence, a Juvenile HormoneEsterase Homolog, and an Alpha Tubulin Homologous Sequence.

Some embodiments comprise an additional component, which may be acarrier, an adjuvant, a solubilizing agent, a suspending agent, adiluent, an oxygen scavenger, an antioxidant, a food material, ananti-contaminant agent, or combinations thereof.

In another embodiment, the additional component(s) may be required forthe application to which the strain or composition is to be utilized.For example, if the strain or composition is to be utilized on, or in,an agricultural product, the additional component(s) may be anagriculturally acceptable carrier, excipient, or diluent. Likewise, ifthe strain or composition is to be utilized on, or in, a foodstuff theadditional component(s) may be an edible carrier, excipient or diluent.

In one aspect, the one or more additional component(s) is a carrier,excipient, or diluent. “Carriers” or “vehicles” mean materials suitablefor compound administration and include any such material known in theart such as, for example, any liquid, gel, solvent, liquid diluent,solubilizer, or the like, which is non-toxic and does not interact withany components of the composition in a deleterious manner.

Examples of nutritionally acceptable carriers include, for example,water, salt solutions, alcohol, silicone, waxes, petroleum jelly,vegetable oils, polyethylene glycols, propylene glycol, liposomes,sugars, gelatin, lactose, amylose, magnesium stearate, talc,surfactants, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Examples of excipients include but are not limited to: microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar, andhigh molecular weight polyethylene glycols.

Examples of diluents include but are not limited to: water, ethanol,propylene glycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes).

The composition or its diluent may also contain chelating agents such asEDTA, citric acid, tartaric acid, etc. Moreover, the composition or itsdiluent may contain active agents selected from fatty acids esters, suchas mono- and diglycerides, non-ionic surfactants, such as polysorbates,phospholipids, etc. Emulsifiers may enhance the stability of thecomposition, especially after dilution.

The bacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof may be used in any suitable form—whether whenalone or when present in a composition. The compositions may beformulated in any suitable way to ensure that the composition comprisesan active compound(s) of interest.

The bacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof compositions thereof may be in the form of adry powder that can be sprinkled on or mixed in with a product. Thecompositions in the form of a dry powder may include an additive such asmicrocrystalline cellulose, gum tragacanth, gelatin, starch, lactose,alginic acid, Primogel, or corn starch (which can be used as adisintegrating agent).

In yet another embodiment, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof compositionsdisclosed herein can be a spray-dried fermentate re-suspended in H₂O toa percentage selected from the following: 0.05-1, 1-3, 3-5, 5-7, 7-10,10-15, 15-20, and greater than 20%. In another embodiment, one or morethan one clarification step(s) can be performed prior to spray-drying.

In one embodiment, the compositions disclosed herein can compriseconcentrated, dried propagules, from the strain disclosed herein. In oneembodiment, compositions can be in the range of 1×10³ to 1×10¹³ CFU/g.

In one embodiment, the compositions disclosed herein can be applied inwet or partially or completely desiccated form or in slurry, gel, orother form.

In at least some embodiments, the compositions disclosed herein can befreeze-dried or lypholized. In at least some embodiments, thecompositions can be mixed with a carrier. The carrier includes but isnot limited to whey, maltodextrin, sucrose, dextrose, limestone (calciumcarbonate), rice hulls, yeast culture, dried starch, clay, and sodiumsilico aluminate. The compositions can also be used with or withoutpreservatives and in concentrated, un-concentrated, or diluted form. Inone embodiment, the compositions can be in the form of a pellet or abiologically pure pellet.

The compositions described herein can be added to one or more carrier.Where used, the carrier(s) and the compositions can be added to a ribbonor paddle mixer and mixed for about 15 minutes, although the timing canbe increased or decreased. The components are blended such that auniform mixture of the culture and carrier(s) is produced. The finalproduct is preferably a dry, flowable powder.

In one embodiment, the compositions may be formulated as a liquid, a drypowder, or a granule. The dry powder or granules may be prepared bymeans known to those skilled in the art, such as, in top-spray fluid bedcoater, in a bottom spray Wurster, or by drum granulation (e.g. highsheer granulation), extrusion, pan coating or in a micro-ingredientsmixer.

In another embodiment, the compositions disclosed herein may be providedas a spray-dried or freeze-dried powder.

In yet another embodiment, the compositions are in a liquid formulation.Such liquid consumption may contain one or more of the following: abuffer, salt, sorbitol, and/or glycerol.

In one embodiment, the compositions disclosed herein may be formulatedwith at least one physiologically acceptable carrier selected from atleast one of maltodextrin, calcined (illite) clay, limestone (calciumcarbonate), cyclodextrin, wheat or a wheat component, sucrose, starch,Na₂SO₄, Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose,propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride,citrate, acetate, phosphate, calcium, metabisulfite, formate andmixtures thereof.

In one embodiment, the compositions disclosed herein may be formulatedby encapsulation technology to improve stability and as a way to protectthe compositions from seed applications. In one embodiment theencapsulation technology may comprise a bead polymer for timed releaseof the compositions over time. In one embodiment, the encapsulatedcompositions may be applied in a separate application of beads in-furrowto the seeds. In another embodiment, the encapsulated compositions maybe co-applied along with seeds simultaneously.

The coating agent usable for the sustained release microparticles of anencapsulation embodiment may be a substance which is useful for coatingthe microgranular form with the substance to be supported thereon. Anycoating agent which can form a coating difficultly permeable for thesupported substance may be used in general, without any particularlimitation. For example, higher saturated fatty acid, wax, thermoplasticresin, thermosetting resin and the like may be used.

Examples of useful higher saturated fatty acid include stearic acid,zinc stearate, stearic acid amide and ethylenebis-stearic acid amide;those of wax include synthetic waxes such as polyethylene wax, carbonwax, Hoechst wax, and fatty acid ester; natural waxes such as carnaubawax, bees wax and Japan wax; and petroleum waxes such as paraffin waxand petrolatum. Examples of thermoplastic resin include polyolefins suchas polyethylene, polypropylene, polybutene and polystyrene; vinylpolymers such as polyvinyl acetate, polyvinyl chloride, polyvinylidenechloride, polyacrylic acid, polymethacrylic acid, polyacrylate andpolymethacrylate; diene polymers such as butadiene polymer, isoprenepolymer, chloroprene polymer, butadiene-styrene copolymer,ethylene-propylene-diene copolymer, styrene-isoprene copolymer,MMA-butadiene copolymer and acrylonitrile-butadiene copolymer;polyolefin copolymers such as ethylene-propylene copolymer,butene-ethylene copolymer, butene-propylene copolymer, ethylene-vinylacetate copolymer, ethylene-acrylic acid copolymer, styreneacrylic acidcopolymer, ethylene-methacrylic acid copolymer, ethylene-methacrylicester copolymer, ethylene-carbon monoxide copolymer, ethylene-vinylacetate-carbon monoxide copolymer, ethylene-vinyl acetate-vinyl chloridecopolymer and ethylene-vinyl acetate-acrylic copolymer; and vinylchloride copolymers such as vinyl chloride-vinyl acetate copolymer andvinylidene chloride-vinyl chloride copolymer. Examples of thermosettingresin include polyurethane resin, epoxy resin, alkyd resin, unsaturatedpolyester resin, phenolic resin, urea-melamine resin, urea resin andsilicone resin. Of those, thermoplastic acrylic ester resin,butadienestyrene copolymer resin, thermosetting polyurethane resin andepoxy resin are preferred, and among the preferred resins, particularlythermosetting polyurethane resin is preferred. These coating agents canbe used either singly or in combination of two or more kinds.

In one embodiment, the compositions may include a seed, a part of aseed, a plant, or a plant part.

All plants, plant parts, seeds or soil may be treated in accordance withthe compositions and methods disclosed herein. The compositionsdisclosed herein may include a plant, a plant part, a seed, a seed part,or soil. The compositions and methods disclosed herein may be applied tothe seed, the plant or plant parts, the fruit, or the soil in which theplants grow.

Some embodiments relate to a method for reducing plant pathogen, pest,or insect damage to a plant or plant part comprising: (a) treating aseed with a composition disclosed herein prior to planting. In anotherembodiment, the method further comprises: (b) treating a plant partobtained from the seed with a composition disclosed herein. Thecomposition used in step (a) may be the same or different than thecomposition used in step (b).

Some embodiments relate to a method for reducing plant pathogen, pest,or insect damage to a plant or plant part comprising: (a) treating thesoil surrounding a seed or plant a bacterial strain disclosed herein, ora progeny, mutant, or variant thereof, a fermentate produced from astrain disclosed herein progeny, mutant, or variant thereof. In anotherembodiment, the method further comprises: (b) treating a plant part witha bacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof disclosed herein. The bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof used in step (a) may be the same or different than a bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof used in step (b).

Some embodiments relate to a method for reducing plant pathogen, pest,or insect damage to a plant or plant part comprising: (a) treating aseed prior to planting with a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed herein.In another embodiment, the method further comprises: (b) treating thesoil surrounding the seed or plant with a bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, a fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereofdisclosed herein. In still another embodiment, the method furthercomprises: (c) treating a plant part of a plant produced from the seedwith a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof disclosed herein. The bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof used in step (a) may be the same or different than thebacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof used in step (b). The bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof used in step (a) may be the same or different than the bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof, used in step (c). The bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, a fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereof usedin step (b) may be the same or different than the bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof used in step (c).

In one embodiment, wild plant species and plant cultivars, or thoseobtained by conventional biological breeding, such as crossing orprotoplast fusion, and parts thereof, can be treated with a bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof disclosed herein. In another embodiment, transgenicplants and plant cultivars obtained by genetic engineering, and plantparts thereof, are treated with a bacterial strain disclosed herein, ora progeny, mutant, or variant thereof, a fermentate produced from astrain disclosed herein progeny, mutant, or variant thereof disclosedherein.

In another embodiment, plants or plant cultivars (obtained by plantbiotechnology methods such as genetic engineering or editing) that maybe treated according to the strains, compositions and methods disclosedherein are herbicide-tolerant plants, i.e. plants made tolerant to oneor more given herbicides. Such plants can be obtained either by geneticmodification, or by selection of plants containing a mutation impartingsuch herbicide tolerance. Herbicide-resistant plants are for exampleglyphosate-tolerant plants, i.e. plants made tolerant to the herbicideglyphosate or salts thereof. Plants can be made tolerant to glyphosatethrough different means. For example, glyphosate-tolerant plants can beobtained by transforming the plant with a gene encoding the enzyme5-enolpyruvylshilcimate-3-phosphate synthase (EPSPS).

Plants or plant cultivars (obtained by plant biotechnology methods suchas genetic engineering or editing) that may also be treated areinsect-resistant genetically modified plants, i.e. plants made resistantto attack by certain target insects. Such plants can be obtained bygenetic transformation, or by selection of plants containing a mutationimparting such insect resistance.

In another embodiment, plants or plant cultivars (obtained by plantbiotechnology methods such as genetic engineering) that may be treatedaccording to the disclosure are tolerant to abiotic stresses. Suchplants can be obtained by genetic transformation, or by selection ofplants containing a mutation imparting such stress resistance.

In another embodiment, plants or plant cultivars (obtained by plantbiotechnology methods such as genetic engineering or editing) that maybe treated according to the disclosure are conventionally bred, bymutagenesis, or genetically engineered to contain a combination or stackof valuable traits, including but not limited to, herbicide tolerance,insect resistance, and abiotic stress tolerance.

The embodiments disclosed herein also apply to plant varieties whichwill be developed, or marketed, in the future and which have thesegenetic traits or traits to be developed in the future.

As used herein, applying a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof to a seed, a plant,or plant part includes contacting the seed, plant, or plant partdirectly and/or indirectly with the bacterial strain disclosed herein,or a progeny, mutant, or variant thereof, a fermentate produced from astrain disclosed herein progeny, mutant, or variant thereof. In oneembodiment, a bacterial strain disclosed herein, or a progeny, mutant,or variant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof may be directly applied as a spray,a rinse, or a powder, or any combination thereof.

As used herein, a spray refers to a mist of liquid particles thatcontain a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof of the present disclosure. In oneembodiment, a spray may be applied to a plant or plant part while aplant or plant part is being grown. In another aspect, a spray may beapplied to a plant or plant part while a plant or plant part is beingfertilized. In another aspect, a spray may be applied to a plant orplant part while a plant or plant part is being harvested. In anotheraspect, a spray may be applied to a plant or plant part after a plant orplant part has been harvested. In another aspect, a spray may be appliedto a plant or plant part while a plant or plant part is being processed.In another aspect, a spray may be applied to a plant or plant part whilea plant or plant part is being packaged. In another aspect, a spray maybe applied to a plant or plant part while a plant or plant part is beingstored.

In another embodiment, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinmay be applied directly to a plant or plant part as a rinse. As usedherein, a rinse is a liquid containing a bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, a fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereofdisclosed herein. Such a rinse may be poured over a plant or plant part.A plant or plant part may also be immersed or submerged in the rinse,then removed and allowed to dry.

In another embodiment, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof may be applied to aplant or plant part and may cover 50% of the surface area of a plantmaterial. In another embodiment, a bacterial strain disclosed herein, ora progeny, mutant, or variant thereof, a fermentate produced from astrain disclosed herein progeny, mutant, or variant thereof may beapplied to a plant or plant part and may cover a percentage of thesurface area of a plant material selected from the group consisting of:from 50% to about 95%, from 60% to about 95%, from 70% to about 95%,from 80% to about 95%, and from 90% to about 95%.

In another aspect, a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof may cover fromabout 20% to about 30%, from about 30% to about 40%, from about 40% toabout 50%, from about 50% to about 60%, from about 60% to about 70%,from about 70% to about 80%, from about 80% to about 90%, from about 90%to about 95%, from about 95% to about 98%, from about 98% to about 99%or 100% of the surface area of a plant or plant part.

In another aspect, a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinmay be applied directly to a plant or plant part as a powder. As usedherein, a powder is a dry or nearly dry bulk solid composed of a largenumber of very fine particles that may flow freely when shaken ortilted. A dry or nearly dry powder composition disclosed hereinpreferably contains a low percentage of water, such as, for example, invarious aspects, less than 5%, less than 2.5%, or less than 1% byweight.

In another aspect, a composition can be applied indirectly to the plantor plant part. For example, a plant or plant part having a bacterialstrain disclosed herein, or a progeny, mutant, or variant thereof, afermentate produced from a strain disclosed herein progeny, mutant, orvariant thereof already applied may be touching a second plant or plantpart so that a bacterial strain disclosed herein, or a progeny, mutant,or variant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof rubs off on a second plant or plantpart. In a further aspect, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof may be appliedusing an applicator. In various aspects, an applicator may include, butis not limited to, a syringe, a sponge, a paper towel, or a cloth, orany combination thereof.

A contacting step may occur while a plant material is being grown, whilea plant or plant part is being fertilized, while a plant or plant partis being harvested, after a plant or plant part has been harvested,while a plant or plant part is being processed, while a plant or plantpart is being packaged, or while a plant or plant part is being storedin warehouse or on the shelf of a store.

In another embodiment, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinmay be a colloidal dispersion. A colloidal dispersion is a type ofchemical mixture where one substance is dispersed evenly throughoutanother. Particles of the dispersed substance are only suspended in themixture, unlike a solution, where they are completely dissolved within.This occurs because the particles in a colloidal dispersion are largerthan in a solution—small enough to be dispersed evenly and maintain ahomogenous appearance, but large enough to scatter light and notdissolve. Colloidal dispersions are an intermediate between homogeneousand heterogeneous mixtures and are sometimes classified as either“homogeneous” or “heterogeneous” based upon their appearance.

In one embodiment, the bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof compositions andmethods disclosed herein are suitable for use with a seed. In anotherembodiment, the bacterial strain disclosed herein, or a progeny, mutant,or variant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof compositions and methods disclosedherein are suitable for use with a seed of one or more of any of theplants recited previously.

In still another embodiment, the bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof compositions andmethods disclosed herein can be used to treat transgenic or genetically,modified or edited seed. A transgenic seed refers to the seed of plantscontaining at least one heterologous gene that allows the expression ofa polypeptide or protein not naturally found in the plant. Theheterologous gene in transgenic seed can originate, for example, frommicroorganisms of the species Bacillus, Rhizobium, Pseudomonas,Serratia, Trichoderma, Clavibacter, Glomus or Gliocladium.

In one embodiment, the seed is treated in a state in which it issufficiently stable so that the treatment does not cause any damage. Ingeneral, treatment of the seed may take place at any point in timebetween harvesting and sowing. In one embodiment, the seed used isseparated from the plant and freed from cobs, shells, stalks, coats,hairs or the flesh of the fruits. Thus, it is possible to use, forexample, seed which has been harvested, cleaned and dried.Alternatively, it is also possible to use seed which, after drying, hasbeen treated, for example, with water and then dried again.

In one embodiment, seed is treated with a bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, a fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereofcompositions and methods disclosed herein in such a way that thegermination of the seed is not adversely affected, or that the resultingplant is not damaged.

In one embodiment, a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof compositionsdisclosed herein may be applied directly to the seed. For example, thebacterial strain disclosed herein, or a progeny, mutant, or variantthereof, fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof compositions disclosed herein may be appliedwithout additional components and without having been diluted.

In another embodiment, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinmay be applied to the seed in the form of a suitable formulation.Suitable formulations and methods for the treatment of seed are known tothe person skilled in the art and are described, for example, in thefollowing documents: U.S. Pat. Nos. 4,272,417 A, 4,245,432 A, 4,808,430A, 5,876,739 A, US 2003/0176428 A1, WO 2002/080675 A1, WO 2002/028186A2.

A bacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof disclosed herein can be converted intocustomary seed dressing formulations, such as solutions, emulsions,suspensions, powders, foams, slurries or other coating materials forseed, and also ULV formulations. These formulations are prepared in aknown manner by mixing A bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinwith customary additives, such as, for example, customary extenders andalso solvents or diluents, colorants, wetting agents, dispersants,emulsifiers, defoamers, preservatives, secondary thickeners, adhesives,gibberellins and water as well.

In another embodiment, suitable colorants that may be present in theseed dressing formulations include all colorants customary for suchpurposes. Use may be made both of pigments, of sparing solubility inwater, and of dyes, which are soluble in water. Examples that may bementioned include the colorants known under the designations RhodamineB, C.I. Pigment Red 112, and C.I. Solvent Red 1.

In another embodiment, suitable wetting agents that may be present inthe seed dressing formulations include all substances that promotewetting and are customary in the formulation of active agrochemicalsubstances. With preference it is possible to usealkylnaphthalene-sulphonates, such as diisopropyl- ordiisobutylnaphthalene-sulphonates.

In still another embodiment, suitable dispersants and/or emulsifiersthat may be present in the seed dressing formulations include allnonionic, anionic, and cationic dispersants that are customary in theformulation of active agrochemical substances. In one embodiment,nonionic or anionic dispersants or mixtures of nonionic or anionicdispersants can be used. In one embodiment, nonionic dispersants includebut are not limited to ethylene oxide-propylene oxide block polymers,alkylphenol polyglycol ethers, and tristyrylphenol polyglycol ethers,and their phosphated or sulphated derivatives.

In still another embodiment, defoamers that may be present in the seeddressing formulations to be used include all foam-inhibiting compoundsthat are customary in the formulation of agrochemically active compoundsincluding but not limited to silicone defoamers, magnesium stearate,silicone emulsions, long-chain alcohols, fatty acids and their salts andalso organofluorine compounds and mixtures thereof.

In still another embodiment, secondary thickeners that may be present inthe seed dressing formulations include all compounds which can be usedfor such purposes in agrochemical compositions, including but notlimited to cellulose derivatives, acrylic acid derivatives,polysaccharides, such as xanthan gum or Veegum, modified clays,phyllosilicates, such as attapulgite and bentonite, and also finelydivided silicic acids.

Suitable adhesives that may be present in the seed dressing formulationsmay include all customary binders which can be used in seed dressings.Polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol and tylosemay be mentioned as being preferred.

In yet another embodiment, seed dressing formulations may be useddirectly or after dilution with water beforehand to treat seed of any ofa very wide variety of types. The seed dressing formulations or theirdilute preparations may also be used to dress seed of transgenic plants.In this context, synergistic effects may also arise in interaction withthe substances formed by expression.

Suitable mixing equipment for treating seed with the seed dressingformulations or the preparations prepared from them by adding waterincludes all mixing equipment that can commonly be used for dressing.The specific procedure adopted when dressing comprises introducing theseed into a mixer, adding the particular desired amount of seed dressingformulation, either as it is or following dilution with waterbeforehand, and carrying out mixing until the formulation is uniformlydistributed on the seed. Optionally, a drying operation follows.

In various embodiments, a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof, can be added tothe plant, plant part, and/or seed at a rate of about 1×10² to 1×10¹³colony forming units (cfu) per seed, including about 1×10³ cfu/seed, orabout 1×10⁴ cfu/seed, 1×10⁵ cfu/seed, or about 1×10⁶ cfu/seed, or about1×10⁷ cfu/seed, or about 1×10⁸ cfu/seed, or about 1×10⁹ cfu/seed, orabout 1×10¹⁰ cfu/seed, or about 1×10¹¹ cfu/seed, or about 1×10¹²cfu/seed, or about 1×10¹³ cfu/seed including about 1×10³ to 1×10⁸cfu/seed about 1×10³ to 1×10⁷ cfu/seed, about 1×10³ to 1×10⁵ cfu/seed,about 1×10³ to 1×10⁶ cfu/seed, about 1×10³ to 1×10⁴ cfu/seed, about1×10³ to 1×10⁹ cfu/seed, about 1×10³ to 1×10¹⁰ cfu/seed, about 1×10³ to1×10¹¹ cfu/seed, about 1×10³ to 1×10¹² cfu/seed, about 1×10 ³ to 1×10¹³cfu/seed, about 1×10⁴ to 1×10⁸ cfu/seed about 1×10⁴ to 1×10⁷ cfu/seed,about 1×10⁴ to 1×10⁵ cfu/seed, about 1×10⁴ to 1×10⁶ cfu/seed, about1×10⁴ to 1×10⁹ cfu/seed, about 1×10⁴ to 1×10¹⁰ cfu/seed, about 1×10¹¹ to1×10⁹ cfu/seed, about 1×10⁴ to 1×10¹² cfu/seed about 1×10⁴ to 1×10¹³cfu/seed, about 1×10⁵ to 1×10⁷ cfu/per seed, about 1×10⁵ to 1×10⁶cfu/per seed, about 1×10⁵ to 1×10⁸ cfu/per seed, about 1×10⁵ to 1×10⁹cfu/per seed, about 1×10⁵ to 1×10¹⁰ cfu/per seed, about 1×10⁵ to 1×10¹¹cfu/per seed, about 1×10⁵ to 1×10¹² cfu/per seed, about 1×10⁵ to 1×10¹³cfu/per seed, about 1×10⁶ to 1×10⁸ cfu/per seed, about 1×10⁶ to 1×10⁷cfu/per seed, about 1×10⁶ to 1×10⁹ cfu/per seed, about 1×10⁶ to 1×10¹⁰cfu/per seed, about 1×10⁶ to 1×10¹¹ cfu/per seed, about 1×10⁶ to 1×10¹²cfu/per seed, about 1×10⁶ to 1×10¹³ cfu/per seed, about 1×10⁷ to 1×10⁸cfu/per seed, about 1×10⁷ to 1×10⁹ cfu/per seed, about 1×10⁷ to 1×10¹⁰cfu/per seed, about 1×10⁷ to 1×10¹¹ cfu/per seed, about 1×10⁷ to 1×10¹²cfu/per seed, about 1×10⁷ to 1×10¹³ cfu/per seed, about 1×10⁸ to 1×10⁹cfu/per seed, about 1×10⁸ to 1×10¹⁰ cfu/per seed, about 1×10⁸ to 1×10¹¹cfu/per seed, about 1×10⁸ to 1×10¹² cfu/per seed, about 1×10⁸ to 1×10¹³cfu/per seed, about 1×10⁹ to 1×10¹⁰ cfu/per seed, about 1×10⁹ to 1×10¹¹cfu/per seed, about 1×10⁹ to 1×10¹² cfu/per seed, about 1×10⁹ to 1×10¹³cfu/per seed, about 1×10¹⁰ to 1×10¹¹ cfu/per seed, about 1×10¹⁰ to1×10¹² cfu/per seed, about 1×10¹⁰ to 1×10¹³ cfu/per seed, about 1×10¹¹¹to 1×10¹² cfu/per seed, about 1×10¹¹ to 1×10¹³ cfu/per seed, and about1×10¹² to 1×10¹³ cfu/per seed. As used herein, the tem “colony formingunit” or “cfu” is a unit capable of growing and producing a colony of amicrobial strain in favorable conditions.

In one embodiment, a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof, may be formulatedas a liquid seed treatment. A seed treatment may comprise at least one abacterial strain disclosed herein, or a progeny, mutant, or variantthereof, a fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof. The seeds are substantially uniformly coatedwith one or more layers of a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof, using conventionalmethods of mixing, spraying or a combination thereof. Application isdone using equipment that accurately, safely, and efficiently appliesseed treatment products to seeds. Such equipment uses various types ofcoating technology such as rotary coaters, drum coaters, fluidized bedtechniques, spouted beds, rotary mists or a combination thereof.

In one embodiment, the application is done via either a spinning“atomizer” disk or a spray nozzle that evenly distributes the seedtreatment onto the seed as it moves through the spray pattern. In yetanother embodiment, the seed is then mixed or tumbled for an additionalperiod of time to achieve additional treatment distribution and drying.The seeds may be primed or unprimed before coating with a compositiondisclosed herein to increase the uniformity of germination andemergence. In an alternative embodiment, a dry powder composition can bemetered onto the moving seed.

In still another embodiment, the seeds may be coated via a continuous orbatch coating process. In a continuous coating process, continuous flowequipment simultaneously meters both the seed flow and the seedtreatment products. A slide gate, cone and orifice, seed wheel, orweight device (belt or diverter) regulates seed flow. Once the seed flowrate through treating equipment is determined, the flow rate of the seedtreatment is calibrated to the seed flow rate in order to deliver thedesired dose to the seed as it flows through the seed treatingequipment. Additionally, a computer system may monitor the seed input tothe coating machine, thereby maintaining a constant flow of theappropriate amount of seed.

In a batch coating process, batch treating equipment weighs out aprescribed amount of seed and places the seed into a closed treatingchamber or bowl where the corresponding of seed treatment is thenapplied. The seed and seed treatment are then mixed to achieve asubstantially uniform coating on each seed. This batch is then dumpedout of the treating chamber in preparation for the treatment of the nextbatch. With computer control systems, this batch process is automatedenabling it to continuously repeat the batch treating process.

A variety of additives can be added to the seed treatments. Binders canbe added and include those composed preferably of an adhesive polymerthat can be natural or synthetic without phytotoxic effect on the seedto be coated. A variety of colorants may be employed, including organicchromophores classified as nitroso, nitro, azo, including monoazo,bisazo, and polyazo, diphenylmethane, triarylmethane, xanthene, methane,acridine, thiazole, thiazine, indamine, indophenol, azine, oxazine,anthraquinone, and phthalocyanine. Other additives that can be addedinclude trace nutrients such as salts of iron, manganese, boron, copper,cobalt, molybdenum, and zinc. A polymer or other dust control agent canbe applied to retain the treatment on the seed surface.

Other conventional seed treatment additives include, but are not limitedto, coating agents, wetting agents, buffering agents, andpolysaccharides. At least one agriculturally acceptable carrier can beadded to the seed treatment formulation such as water, solids or drypowders. The dry powders can be derived from a variety of materials suchas wood barks, calcium carbonate, gypsum, vermiculite, talc, humus,activated charcoal, and various phosphorous compounds.

In one embodiment, the seed coating can comprise of at least one filler,which is an organic or inorganic, natural or synthetic component withwhich a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof described herein is combined tofacilitate its application onto the seed. In one embodiment, the filleris an inert solid such as clays, natural or synthetic silicates, silica,resins, waxes, solid fertilizers (for example ammonium salts), naturalsoil minerals, such as kaolins, clays, talc, lime, quartz, attapulgite,montmorillonite, bentonite, or diatomaceous earths, or syntheticminerals, such as silica, alumina, or silicates, in particular aluminumor magnesium silicates.

In one embodiment, a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinmay be formulated by encapsulation technology to improve fungal sporestability and as a way to protect the fungal spores from seed appliedfungicides. In one embodiment the encapsulation technology may comprisea bead polymer for timed release of fungal spores over time. In oneembodiment, the encapsulation technology may comprise a zeolitematerial. In one embodiment, an encapsulated bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereof maybe applied in a separate application of beads in-furrow to the seeds. Inanother embodiment, the encapsulated bacterial strain disclosed herein,or a progeny, mutant, or variant thereof, fermentate produced from astrain disclosed herein progeny, mutant, or variant thereof may beco-applied along with seeds simultaneously.

Insect resistance management (IRM) is the term used to describepractices aimed at reducing the potential for insect pests to becomeresistant to an insect management tactic. IRM maintenance of Bt(Bacillus thuringiensis) derived pesticidal proteins, other pesticidalproteins, a chemical, a biological agent, or other biologicals, is ofgreat importance because of the threat insect resistance poses to thefuture use of pesticidal plant-incorporated protectants and insecticidaltrait technology as a whole. Specific IRM strategies, such as the refugestrategy, mitigate insect resistance to specific insecticidal proteinsproduced in corn, soybean, cotton, and other crops. However, suchstrategies result in portions of crops being left susceptible to one ormore pests in order to ensure that non-resistant insects develop andbecome available to mate with any resistant pests produced in protectedcrops. Accordingly, from a farmer/producer's perspective, it is highlydesirable to have as small a refuge as possible and yet still manageinsect resistance, in order that the greatest yield be obtained whilestill maintaining the efficacy of the pest control method used, whetherBt, a different pesticidal protein, chemical, biological agent or otherbiologicals, some other method, or combinations thereof.

Another strategy to reduce the need for refuge is the pyramiding oftraits with different modes of action against a target insect pest. Forexample, Bt toxins that have different modes of action pyramided in onetransgenic plant are able to have reduced refuge requirements due toreduced resistance risk. Different modes of action in a pyramidcombination also extend the durability of each trait, as resistance isslower to develop to each trait.

Currently, the size, placement, and management of the refuge are oftenconsidered critical to the success of refuge strategies to mitigateinsect resistance to the Bt/pesticidal trait produced in corn, cotton,soybean, and other crops. Because of the decrease in yield in refugeplanting areas, some farmers choose to eschew the refuge requirements,and others do not follow the size and/or placement requirements. Theseissues result in either no refuge or a less effective refuge, and acorresponding risk of the increase in the development of resistancepests.

Accordingly, there remains a need for methods for managing pestresistance in a plot of pest resistant crop plants. It would be usefulto provide an improved method for the protection of plants, especiallycorn or other crop plants, from feeding damage by pests. It would beparticularly useful if such a method would reduce the requiredapplication rate of conventional chemical pesticides, and also if itwould limit the number of separate field operations that were requiredfor crop planting and cultivation. In addition, it would be useful tohave a method of deploying a biocontrol agent that increases thedurability of an insecticidal trait or increases the efficacy of manyresistance management strategies.

One embodiment relates to a method of reducing or preventing theresistance of pests to a plant pesticidal composition comprisingproviding a plant protection composition, such as a Bt pesticidalprotein, a transgenic pesticidal protein, other pesticidal proteins,chemical pesticides, or pesticidal biological entomopathogens, to aplant and/or plant part or a planted area or insecticidal trait andproviding a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof described herein to the plant and/orplant part or planted area. Another embodiment relates to a method ofreducing or preventing the resistance to a plant insecticidal traitcomprising providing or contacting a plant with a bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof described herein.

A further embodiment relates to a method of increasing the durability ofplant pest compositions comprising providing a plant protectioncomposition to a plant or planted area, and providing a bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof described herein to the plant or planted area, wherein thebacterial strain disclosed herein, or a progeny, mutant, or variantthereof, fermentate produced from a strain disclosed herein progeny,mutant, or variant thereof described herein have a different mode ofaction than the plant protection composition.

In a still further embodiment, the refuge required may be reduced oreliminated by the presence of a bacterial strain disclosed herein, or aprogeny, mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof described hereinapplied to the non-refuge plants. In another embodiment, the refuge mayinclude a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof described herein as a spray, bait,or as a different mode of action.

In one embodiment, a composition comprises a bacterial strain disclosedherein, or a progeny, mutant, or variant thereof, a fermentate producedfrom a strain disclosed herein progeny, mutant, or variant thereofdisclosed herein and a non-Bt insecticidal trait increases resistance toa pathogen, pest, or insect. In another embodiment, the non-Btinsecticidal trait comprises a plant-derived insecticidal protein, abacterial/archeal-derived insecticidal protein not from a Bt (such as aPseudomonas insecticidal protein), an animal-derived insecticidalprotein, or a silencing element. In another embodiment, a compositioncomprising a bacterial strain disclosed herein, or a progeny, mutant, orvariant thereof, a fermentate produced from a strain disclosed hereinprogeny, mutant, or variant thereof disclosed herein and a non-Btinsecticidal trait increases durability of the non-Bt insecticidaltrait. In another embodiment, the non-Bt insecticidal trait comprises aPIP-72 polypeptide of PCT Serial Number PCT/US14/55128. In anotherembodiment, the non-Bt insecticidal trait comprises a polynucleotidesilencing elements targeting RyanR (DvSSJ) (US Patent Applicationpublication 2014/0275208). In another embodiment, the non-Btinsecticidal trait comprises a polynucleotide silencing elementstargeting RyanR (DvSSJ) (US Patent Application publication 2014/0275208,herein incorporated by reference in its entirety) and a PIP-72polypeptide of PCT Serial Number PCT/US14/55128, herein incorporated byreference in its entirety.

In another embodiment, a composition comprising a bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof disclosed herein and a fungal entomopathogen disclosed in U.S.Pat. No. 9,993,006, herein incorporated by reference in its entirety.

In some embodiments, a composition comprises a bacterial straindisclosed herein, or a progeny, mutant, or variant thereof, a fermentateproduced from a strain disclosed herein progeny, mutant, or variantthereof disclosed herein and a Bt insecticidal trait that increasesresistance to a pathogen, pest, or insect. A Bt insecticidal trait mayhave activity to Coleopteran, Lepidopteran, or Hemipteran plant pests.The compositions disclosed herein may provide to a plant or plant partadditive or synergistic resistance to a pathogen, pest, or insect plantin combination with a Bt insecticidal trait. In one embodiment, acomposition comprises a bacterial strain disclosed herein, or a progeny,mutant, or variant thereof, a fermentate produced from a straindisclosed herein progeny, mutant, or variant thereof disclosed hereinand a Bt insecticidal trait, wherein the Bt insecticidal trait comprisesa Cry3B toxin disclosed in U.S. Pat. Nos. 8,101,826, 6,551,962,6,586,365, 6,593,273, and PCT Publication WO 2000/011185, a mCry3B toxindisclosed in U.S. Pat. Nos. 8,269,069, and 8,513,492, a mCry3A toxindisclosed in U.S. Pat. Nos. 8,269,069, 7,276,583 and 8,759,620, or aCry34/35 toxin disclosed in U.S. Pat. Nos. 7,309,785, 7,524,810,7,985,893, 7,939,651 and 6,548,291, and transgenic events containingthese Bt insecticidal toxins and other Coleopteran active Btinsecticidal traits for example, event MON863 disclosed in U.S. Pat. No.7,705,216, event MIR604 disclosed in U.S. Pat. No. 8,884,102, event 5307disclosed in U.S. Pat. No. 9,133,474, event DAS-59122 disclosed in U.S.Pat. No. 7,875,429, event DP-4114 disclosed in U.S. Pat. No. 8,575,434,event MON87411 disclosed in US Published Patent Application Number2013/0340111, and event MON88017 disclosed in U.S. Pat. No. 8,686,230all of which are incorporated herein by reference. All publications,patents and patent applications mentioned in the specification indicatethe level of those skilled in the art to which this disclosure pertains.All publications, patents and patent applications are incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

IPD126 Proteins and Variants and Fragments Thereof

IPD126 polypeptides are encompassed by the disclosure as set forth inSEQ ID NOs: 19-36. “IPD126 polypeptide,” and “IPD126 protein” as usedherein interchangeably refers to a polypeptide(s) having insecticidalactivity including but not limited to insecticidal activity against oneor more insect pests of the Lepidoptera, Hemiptera, and/or Coleopteraorders. A variety of IPD126 polypeptides are contemplated.

“Sufficiently identical” is used herein to refer to an amino acidsequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. Inone embodiment the IPD126 polypeptide has at least about 40%, 45%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity comparedto any one of SEQ ID NOs: 19-36. The term “about” when used herein incontext with percent sequence identity means +/−1.0%.

A “recombinant protein” is used herein to refer to a protein that is nolonger in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to anIPD126 polypeptide and that exhibit insecticidal activity. “Fragments”or “biologically active portions” of IPD126 polypeptides includesfragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in any one of SEQ ID NOs: 19-36 whereinthe IPD126 polypeptide has insecticidal activity. Such biologicallyactive portions can be prepared by recombinant techniques and evaluatedfor insecticidal activity.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identical to the parental aminoacid sequence.

In some embodiments an IPD126 polypeptide comprises an amino acidsequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity to the full length or a fragmentof the amino acid sequence of any one of SEQ ID NOs: 19-36, wherein theIPD126 polypeptide has insecticidal activity.

In some embodiments an IPD126 polypeptide comprises an amino acidsequence of any one or more of SEQ ID NOS: 19-36 having 1, 2, 3, 4, 5,6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 ormore amino acid substitutions compared to the amino acid at thecorresponding position of any one or more of the respective SEQ ID NOS:19-36.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of an IPD126 polypeptide can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis, such as for example site-specificdouble strand break technology, and/or in directed evolution. In someaspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess a desired pesticidal activity. However, it is understood thatthe ability of an IPD126 polypeptide to confer pesticidal activity orother polypeptide physical property may be improved or altered by theuse of such techniques upon the compositions of this disclosure.

Conservative amino acid substitutions may be made at one or morepredicted nonessential amino acid residues. A “nonessential” amino acidresidue is a residue that can be altered from the wild-type sequence ofan IPD126 polypeptide without altering the biological activity. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include: amino acids with basicside chains (e.g., lysine, arginine, histidine); acidic side chains(e.g., aspartic acid, glutamic acid); polar, negatively charged residuesand their amides (e.g., aspartic acid, asparagine, glutamic, acid,glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentIPD126 polypeptide coding regions can be used to create a new IPD126polypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; and U.S. Pat. Nos.5,605,793 and 5,837,458.

In some embodiments, chimeric polypeptides are provided comprisingregions of at least two different IPD126 polypeptides selected from anyone or more of SEQ ID NOS: 19-36.

Nucleic Acid Molecules, and Variants and Fragments Thereof

Isolated or recombinant nucleic acid molecules comprising nucleic acidsequences encoding IPD126 polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify nucleic acid molecules encodingproteins with regions of sequence homology are provided. As used herein,the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences (preferably protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, therecombinant nucleic acid molecules encoding IPD126 polypeptides cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleic acid sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments an isolated nucleic acid molecule encoding IPD126polypeptides has one or more change in the nucleic acid sequencecompared to the native or genomic nucleic acid sequence. In someembodiments the change in the native or genomic nucleic acid sequenceincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; changes in the nucleic acidsequence due to the amino acid substitution, insertion, deletion and/oraddition compared to the native or genomic sequence; removal of one ormore intron; deletion of one or more upstream or downstream regulatoryregions; and deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence. In some embodiments the nucleicacid molecule encoding an IPD126 polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode IPD126 polypeptides or relatedproteins are contemplated. Such polynucleotides are useful forproduction of IPD126 polypeptides in host cells when operably linked toa suitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encodeIPD126 polypeptides or related proteins.

The polynucleotides of any one or more of SEQ ID NOS: 1-18, can be usedto express IPD126 polypeptides in recombinant bacterial hosts thatinclude but are not limited to Agrobacterium, Bacillus, Escherichia,Salmonella, Lysinibacillus, Acetobacter, Pseudomonas and Rhizobiumbacterial host cells. The polynucleotides are also useful as probes forisolating homologous or substantially homologous polynucleotidesencoding IPD126 polypeptides or related proteins. Such probes can beused to identify homologous or substantially homologous polynucleotides,or portions thereof, derived from Bacillus thurengiensis.

Polynucleotides encoding IPD126 polypeptides can also be synthesized denovo from an IPD126 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from an IPD126 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif) can beused to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of IPD126 polypeptide sequencesthat can be used to obtain corresponding nucleotide encoding sequencesinclude, but are not limited to the IPD126 polypeptides of SEQ ID NOS:19-36. Furthermore, synthetic IPD126 polynucleotide sequences of thedisclosure can be designed so that they will be expressed in plants.

In some embodiments the nucleic acid molecule encoding a IPD126polypeptide is a polynucleotide having the sequence set forth in any oneof SEQ ID NOS: 1-18, and variants, fragments and complements thereof.“Complement” is used herein to refer to a nucleic acid sequence that issufficiently complementary to a given nucleic acid sequence such that itcan hybridize to the given nucleic acid sequence to thereby form astable duplex. “Polynucleotide sequence variants” is used herein torefer to a nucleic acid sequence that except for the degeneracy of thegenetic code encodes the same polypeptide.

In some embodiments the nucleic acid molecule encoding the IPD126polypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” or “non-genomic polynucleotide” refers to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; optimization of the nucleic acidsequence for expression in plants; changes in the nucleic acid sequenceto introduce at least one amino acid substitution, insertion, deletionand/or addition compared to the native or genomic sequence; removal ofone or more intron associated with the genomic nucleic acid sequence;insertion of one or more heterologous introns; deletion of one or moreupstream or downstream regulatory regions associated with the genomicnucleic acid sequence; insertion of one or more heterologous upstream ordownstream regulatory regions; deletion of the 5′ and/or 3′ untranslatedregion associated with the genomic nucleic acid sequence; insertion of aheterologous 5′ and/or 3′ untranslated region; and modification of apolyadenylation site. In some embodiments the non-genomic nucleic acidmolecule is a synthetic nucleic acid sequence.

In some embodiments the nucleic acid molecule encoding a IPD126polypeptide disclosed herein is a non-genomic polynucleotide having anucleotide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater sequence identity, to the nucleic acid sequence of anyone of SEQ ID NOS: 1-18, wherein the IPD126 polypeptide has insecticidalactivity.

In some embodiments the nucleic acid molecule encodes an IPD126polypeptide variant comprising one or more amino acid substitutions tothe amino acid sequence of any one of SEQ ID NOS: 19-36.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding IPD126 polypeptides are also encompassed by theembodiments. “nucleotide fragment” as used herein refers to a portion ofthe nucleic acid sequence encoding an IPD126 polypeptide. A nucleotidefragment of a nucleic acid sequence may encode a biologically activeportion of an IPD126 polypeptide or it may be a fragment that can beused as a hybridization probe or PCR primer using methods disclosedbelow. Nucleic acid molecules that are fragments of a nucleic acidsequence encoding an IPD126 polypeptide comprise at least about 150,180, 210, 240, 270, 300, 330, 360, 400, 450, or 500 contiguousnucleotides or up to the number of nucleotides present in a full-lengthnucleic acid sequence encoding an IPD126 polypeptide disclosed herein,depending upon the intended use. “Contiguous nucleotides” is used hereinto refer to nucleotide residues that are immediately adjacent to oneanother. Fragments of the nucleic acid sequences of the embodiments willencode protein fragments that retain the biological activity of theIPD126 polypeptide and, hence, retain insecticidal activity. “Retainsinsecticidal activity” is used herein to refer to a polypeptide havingat least about 10%, at least about 30%, at least about 50%, at leastabout 70%, 80%, 90%, 95% or higher of the insecticidal activity of anyone of the full-length IPD126 polypeptides set forth in SEQ ID NOS:19-36. In some embodiments, the insecticidal activity is against aLepidopteran species. In one embodiment, the insecticidal activity isagainst a Coleopteran species. In some embodiments, the insecticidalactivity is against one or more insect pests of the corn rootwormcomplex: western corn rootworm, Diabrotica virgifera; northern cornrootworm, D. barberi: Southern corn rootworm or spotted cucumber beetle;Diabrotica undecimpunctata howardi, Diabrotica speciosa, and the Mexicancorn rootworm, D. virgifera zeae. In one embodiment, the insecticidalactivity is against a Diabrotica species.

In some embodiments the IPD126 polypeptide is encoded by a nucleic acidsequence sufficiently homologous to any one of the nucleic acidsequences of SEQ ID NOS: 1-18.

“Percent (%) sequence identity” with respect to a reference sequence(subject) is determined as the percentage of amino acid residues ornucleotides in a candidate sequence (query) that are identical with therespective amino acid residues or nucleotides in the reference sequence,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyamino acid conservative substitutions as part of the sequence identity.Alignment for purposes of determining percent sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (e.g., percentidentity of query sequence=number of identical positions between queryand subject sequences/total number of positions of query sequence×100).

In some embodiments an IPD126 polynucleotide encodes an IPD126polypeptide comprising an amino acid sequence having at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence identity across the entirelength of the amino acid sequence of any one of SEQ ID NOS: 19-36.

In some embodiments polynucleotides are provided encoding chimericpolypeptides comprising regions of at least two different IPD126polypeptides of the disclosure.

The embodiments also encompass nucleic acid molecules encoding IPD126polypeptide variants. “Variants” of the IPD126 polypeptide encodingnucleic acid sequences include those sequences that encode the IPD126polypeptides disclosed herein but that differ conservatively because ofthe degeneracy of the genetic code as well as those that aresufficiently identical as discussed above. Naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleic acid sequences alsoinclude synthetically derived nucleic acid sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the IPD126 polypeptides disclosed as discussed below.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the IPD126 polypeptides disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding IPD126 polypeptides of the present disclosure exist.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded IPD126 polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known.Methods for producing a variant of any nucleic acid listed hereincomprising recursively recombining such polynucleotide with a second (ormore) polynucleotide, thus forming a library of variant polynucleotidesare also embodiments of the disclosure, as are the libraries produced,the cells comprising the libraries and any recombinant polynucleotideproduced by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from a bacterial source. In this manner, methodssuch as PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences set forthherein. Sequences that are selected based on their sequence identity tothe entire sequences set forth herein or to fragments thereof areencompassed by the embodiments. Such sequences include sequences thatare orthologs of the disclosed sequences. The term “orthologs” refers togenes derived from a common ancestral gene and which are found indifferent species as a result of speciation. Genes found in differentspecies are considered orthologs when their nucleotide sequences and/ortheir encoded protein sequences share substantial identity as definedelsewhere herein. Functions of orthologs are often highly conservedamong species.

To identify potential IPD126 polypeptides from bacterium collections,the bacterial cell lysates can be screened with antibodies generatedagainst IPD126 using Western blotting and/or ELISA methods. This type ofassay can be performed in a high throughput fashion. Positive samplescan be further analyzed by various techniques such as antibody basedprotein purification and identification. Methods of generatingantibodies are well known in the art as discussed infra.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the IPD126 polypeptide-encoding nucleic acidsequences disclosed herein. Degenerate primers designed on the basis ofconserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequences encoding IPD126 polypeptides ofthe disclosure or a fragment or variant thereof. Methods for thepreparation of probes for hybridization and stringency conditions aregenerally known in the art and are disclosed in Sambrook and Russell,(2001), supra, herein incorporated by reference.

Antibodies

Antibodies to an IPD126 polypeptide of the embodiments or to variants orfragments thereof are also encompassed. The antibodies of the disclosureinclude polyclonal and monoclonal antibodies as well as fragmentsthereof which retain their ability to bind to an IPD126 polypeptide. Anantibody, monoclonal antibody or fragment thereof is said to be capableof binding a molecule if it is capable of specifically reacting with themolecule to thereby bind the molecule to the antibody, monoclonalantibody or fragment thereof. The term “antibody” (Ab) or “monoclonalantibody” (Mab) is meant to include intact molecules as well asfragments or binding regions or domains thereof (such as, for example,Fab and F(ab).sub.2 fragments) which are capable of binding hapten. Suchfragments are typically produced by proteolytic cleavage, such as papainor pepsin. Alternatively, hapten-binding fragments can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry. Methods for the preparation of the antibodies ofthe present disclosure are generally known in the art. For example, see,Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) ColdSpring Harbor Laboratory, N.Y. (1988), as well as the references citedtherein. Standard reference works setting forth the general principlesof immunology include: Klein, J. Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al.,Monoclonal Antibodies, Hybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, N.Y. (1980) and Campbell, “Monoclonal AntibodyTechnology,” In Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984).See also, U.S. Pat. Nos. 4,196,265; 4,609,893; 4,713,325; 4,714,681;4,716,111; 4,716,117 and 4,720,459. Antibodies against IPD126polypeptides or antigen-binding portions thereof can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, for example the standard somatic cell hybridizationtechnique of Kohler and Milstein, (1975) Nature 256:495. Othertechniques for producing monoclonal antibody can also be employed suchas viral or oncogenic transformation of B lymphocytes. An animal systemfor preparing hybridomas is a murine system. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known. The antibody and monoclonal antibodies of thedisclosure can be prepared by utilizing an IPD126 polypeptide asantigens.

A kit for detecting the presence of an IPD126 polypeptide or detectingthe presence of a nucleotide sequence encoding an IPD126 polypeptide ina sample is provided. In one embodiment, the kit provides antibody-basedreagents for detecting the presence of an IPD126 polypeptide in a tissuesample. In another embodiment, the kit provides labeled nucleic acidprobes useful for detecting the presence of one or more polynucleotidesencoding an IPD126 polypeptide. The kit is provided along withappropriate reagents and controls for carrying out a detection method,as well as instructions for use of the kit.

Receptor Identification and Isolation

Receptors to the IPD126 polypeptides of the embodiments or to variantsor fragments thereof are also encompassed. Methods for identifyingreceptors are known in the art (see, Hofmann, et. al., (1988) Eur. J.Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282) andcan be employed to identify and isolate the receptor that recognizes theIPD126 polypeptide using the brush-border membrane vesicles fromsusceptible insects. In addition to the radioactive labeling methodlisted in the cited literatures, an IPD126 polypeptide can be labeledwith fluorescent dye and other common labels such as streptavidin.Brush-border membrane vesicles (BBMV) of susceptible insects such assoybean looper and stink bugs can be prepared according to the protocolslisted in the references of Hofmann and Gill above and separated onSDS-PAGE gel and blotted on suitable membrane. Labeled IPD126polypeptide can be incubated with blotted membrane of BBMV and labeledIPD126 polypeptide can be identified with the labeled reporters.Identification of protein band(s) that interact with the IPD126polypeptide can be detected by N-terminal amino acid gas phasesequencing or mass spectrometry based protein identification method(Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biologypublished by John Wiley & Son Inc). Once the protein is identified, thecorresponding gene can be cloned from genomic DNA or cDNA library of thesusceptible insects and binding affinity can be measured directly withthe IPD126 polypeptide. Receptor function for insecticidal activity bythe IPD126 polypeptide can be verified by RNAi type of gene knock outmethod (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructs,particularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides, may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the IPD126 polypeptide gene sequence of the disclosureto be under the transcriptional regulation of the regulatory regions.The DNA construct may additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter or any other nucleotide or amino acid sequence is “foreign” or“heterologous” to the sequence of the embodiments, it is intended thatthe nucleotide or amino acid sequence is not the native or naturallyoccurring promoter or nucleotide sequence for the operably linkedsequence of the embodiments. As used herein, a chimeric gene comprises acoding sequence operably linked to a transcription initiation regionthat is heterologous to the coding sequence. Where the promoter is anative or natural sequence, the expression of the operably linkedsequence is altered from the wild-type expression, which results in analteration in phenotype.

In some embodiments the DNA construct comprises a polynucleotideencoding an IPD126 polypeptide of the embodiments. In some embodimentsthe DNA construct comprises a polynucleotide encoding a fusion proteincomprising an IPD126 polypeptide of the embodiments.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464)), the omega enhancer or theomega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNAed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990)EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may alsobe used. The above list of transcriptional enhancers is not meant to belimiting. Any appropriate transcriptional enhancer can be used in theembodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred usage. For example, although nucleic acid sequences ofthe embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific preferences and GC content preferences of monocotyledons ordicotyledons as these preferences have been shown to differ (Murray etal. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred fora particular amino acid may be derived from known gene sequences frommaize. Maize usage for 28 genes from maize plants is listed in Table 4of Murray, et al., supra. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep37:677-684, 2010, herein incorporated by reference. A Zea maize usagetable can be also found at kazusa.or.jp//cgi-bin/show.cgi?species=4577,which can be accessed using the www prefix. A Glycine max usage tablecan be found atkazusa.or.jp//cgi-bin/show.cgi?species=3847&aa=1&style=N, which can beaccessed using the www prefix.

In some embodiments the recombinant nucleic acid molecule encoding anIPD126 polypeptide has maize optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell.Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide(s) or polypeptide(s) gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide(s) or polypeptide(s) into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Led transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger,et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987)Particulate Science and Technology 5:27-37 (onion); Christou, et al.,(1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens).

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the IPD126 polynucleotide or variants and fragmentsthereof directly into the plant or the introduction of the IPD126polypeptide transcript into the plant. Such methods include, forexample, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al, (1994) Proc. Natl. Acad. Sci.91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784. Alternatively, the IPD126 polynucleotide can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector system and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle-bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethylimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853. Briefly, the polynucleotide of the embodiments can becontained in transfer cassette flanked by two non-identicalrecombination sites. The transfer cassette is introduced into a planthave stably incorporated into its genome a target site which is flankedby two non-identical recombination sites that correspond to the sites ofthe transfer cassette. An appropriate recombinase is provided and thetransfer cassette is integrated at the target site. The polynucleotideof interest is thereby integrated at a specific chromosomal position inthe plant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al, (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa triviahs); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinelia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, favabean, lentils, chickpea, etc.

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In Northern blotanalysis, RNA is isolated from specific tissues of transformant,fractionated in a formaldehyde agarose gel, and blotted onto a nylonfilter according to standard procedures that are routinely used in theart (Sambrook and Russell, (2001) supra). Expression of RNA encoded bythe pesticidal gene is then tested by hybridizing the filter to aradioactive probe derived from a pesticidal gene, by methods known inthe art (Sambrook and Russell, (2001) supra). Western blot, biochemicalassays and the like may be carried out on the transgenic plants toconfirm the presence of protein encoded by the pesticidal gene bystandard procedures (Sambrook and Russell, 2001, supra) using antibodiesthat bind to one or more epitopes present on the IPD126 polypeptide.

Methods to Introduce Genome Editing Technologies into Plants

In some embodiments, the disclosed IPD126 polynucleotide compositionscan be introduced into the genome of a plant using genome editingtechnologies, or previously introduced IPD126 polynucleotides in thegenome of a plant may be edited using genome editing technologies. Forexample, the disclosed polynucleotides can be introduced into a desiredlocation in the genome of a plant through the use of double-strandedbreak technologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. For example, the disclosed polynucleotides canbe introduced into a desired location in a genome using a CRISPR-Cassystem, for the purpose of site-specific insertion. The desired locationin a plant genome can be any desired target site for insertion, such asa genomic region amenable for breeding or may be a target site locatedin a genomic window with an existing trait of interest. Existing traitsof interest could be either an endogenous trait or a previouslyintroduced trait.

In some embodiments, where the disclosed IPD126 polynucleotide haspreviously been introduced into a genome, genome editing technologiesmay be used to alter or modify the introduced polynucleotide sequence.Site specific modifications that can be introduced into the disclosedIPD126 polynucleotide compositions include those produced using anymethod for introducing site specific modification, including, but notlimited to, through the use of gene repair oligonucleotides (e.g. USPublication 2013/0019349), or through the use of double-stranded breaktechnologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. Such technologies can be used to modify thepreviously introduced polynucleotide through the insertion, deletion orsubstitution of nucleotides within the introduced polynucleotide.Alternatively, double-stranded break technologies can be used to addadditional nucleotide sequences to the introduced polynucleotide.Additional sequences that may be added include, additional expressionelements, such as enhancer and promoter sequences. In anotherembodiment, genome editing technologies may be used to positionadditional insecticidally-active proteins in proximity to the disclosedIPD126 polynucleotide compositions disclosed herein within the genome ofa plant, in order to generate molecular stacks of insecticidally-activeproteins.

An “altered target site,” “altered target sequence.” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments, one or more of the polynucleotides encoding theIPD126 polypeptide(s) disclosed herein, alone or stacked with one ormore additional insect resistance traits can be stacked with one or moreadditional input traits (e.g., herbicide resistance, fungal resistance,virus resistance, stress tolerance, disease resistance, male sterility,stalk strength, and the like) or output traits (e.g., increased yield,modified starches, improved oil profile, balanced amino acids, highlysine or methionine, increased digestibility, improved fiber quality,drought resistance, and the like). Thus, the polynucleotide embodimentscan be used to provide a complete agronomic package of improved cropquality with the ability to flexibly and cost effectively control anynumber of agronomic pests.

Transgenes useful for stacking include but are not limited to:transgenes that confer resistance to an herbicide; transgenes thatconfer or contribute to an altered grain characteristic; genes thatcontrol male-sterility; genes that create a site for site specific dnaintegration; genes that affect abiotic stress resistance; genes thatconfer increased yield genes that confer plant digestibility; andtransgenes that confer resistance to insects or disease.

Examples of transgenes that confer resistance to insects include genesencoding a Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,(1986) Gene 48:109, who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC® Accession Numbers40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillusthuringiensis transgenes being genetically engineered are given in thefollowing patents and patent applications: U.S. Pat. Nos. 5,188,960;5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594, 6,063,597,6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826, 7,105,332;7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736,7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862, 7,605,304,7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846, 7,858,849 and WO1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581 and WO1997/40162.

Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcaligenes(Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al.,(2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteinsfrom Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010)TheOpen Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied andEnvir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and 6,379,946; aPIP-1 polypeptide of U.S. Pat. No. 9,688,730; an AfIP-1A and/or AfIP-1Bpolypeptide of U.S. Pat. No. 9,475,847; a PIP-47 polypeptide of USPublication Number US20160186204; an IPD045 polypeptide, an IPD064polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077polypeptide of PCT Publication Number WO 2016/114973; an IPD080polypeptide of PCT Serial Number PCT/US17/56517; an IPD078 polypeptide,an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, anIPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide ofSerial Number PCT/US17/54160; PIP-72 polypeptide of US PatentPublication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65polypeptide of US Publication Number US20170166921; an IPD098polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109polypeptide of U.S. Ser. No. 62/521,084; a PtIP-83 polypeptide of USPublication Number US20160347799; a PtIP-96 polypeptide of USPublication Number US20170233440; an IPD079 polypeptide of PCTPublication Number WO2017/23486; an IPD082 polypeptide of PCTPublication Number WO 2017/105987, an IPD090 polypeptide of SerialNumber PCT/US17/30602, an IPD093 polypeptide of U.S. Ser. No.62/434,020; an IPD103 polypeptide of Serial Number PCT/US17/39376; anIPD101 polypeptide of U.S. Ser. No. 62/438,179; an IPD121 polypeptide ofUS Serial Number U.S. 62/508,514, and δ-endotoxins including, but notlimited to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9,Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19,Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29,Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39,Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50,Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60,Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70,Cry71, and Cry72 classes of δ-endotoxin genes and the B. thuringiensiscytolytic Cyt1 and Cyt2 genes.

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of Cryproteins such as Cry1A) of U.S. Pat. Nos. 8,304,604 and 8,304,605, Cry1Bof U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No.6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/Fchimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3Aprotein including but not limited to an engineered hybrid insecticidalprotein (eHIP) created by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins (US PatentApplication Publication Number 2010/0017914); a Cry4 protein; a Cry5protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736,7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; aCry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D,Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008)Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33and CryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330,6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of USPatent Publication Number 2006/0191034, 2012/0278954, and PCTPublication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos.6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry51 protein, aCry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No.8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No.7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891; AXMI-010of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965;AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 ofUS20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019,AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023,AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and relatedproteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z andAXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227,AXMI228, AXMI229, AXMI230, and AXMI231 of WO11/103247; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211;AXMI-066 and AXMI-076 of US2009/0144852; AXMI128, AXMI130, AXMI131,AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148,AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158,AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171,AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543;and Cry proteins such as Cry1A and Cry3A having modified proteolyticsites of U.S. Pat. No. 8,319,019; and a Cry1Ac, Cry2Aa and Cry1Ca toxinprotein from Bacillus thuringiensis strain VBTS 2528 of US PatentApplication Publication Number 2011/0064710. Other Cry proteins are wellknown to one skilled in the art (see, Crickmore, et al., “Bacillusthuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/which can be accessed on theworld-wide web using the “www” prefix). The insecticidal activity of Cryproteins is well known to one skilled in the art (for review, see, vanFrannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to Cry1Ac,Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab,Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c andCBI-Bt have received regulatory approval (see, Sanahuja, (2011) PlantBiotech Journal 9:283-300 and the CERA (2010) GM Crop Database Centerfor Environmental Risk Assessment (CERA), ILSI Research Foundation,Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database whichcan be accessed on the world-wide web using the “www” prefix). More thanone pesticidal proteins well known to one skilled in the art can also beexpressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE &Cry1F (US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa,Cry1I or Cry1E (US2012/0324605). Pesticidal proteins also includeinsecticidal lipases including lipid acyl hydrolases of U.S. Pat. No.7,491,869, and cholesterol oxidases such as from Streptomyces (Purcellet al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidalproteins also include VIP (vegetative insecticidal proteins) toxins ofU.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686,and 8,237,020, and the like. Other VIP proteins are well known to oneskilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

Further transgenes that confer resistance to insects may down-regulationof expression of target genes in insect pest species by interferingribonucleic acid (RNA) molecules through RNA interference. RNAinterference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). RNAitransgenes may include but are not limited to expression of dsRNA,siRNA, miRNA, iRNA, antisense RNA, or sense RNA molecules thatdown-regulate expression of target genes in insect pests. PCTPublication WO 2007/074405 describes methods of inhibiting expression oftarget genes in invertebrate pests including Colorado potato beetle. PCTPublication WO 2005/110068 describes methods of inhibiting expression oftarget genes in invertebrate pests including in particular Western cornrootworm as a means to control insect infestation. Furthermore, PCTPublication WO 2009/091864 describes compositions and methods for thesuppression of target genes from insect pest species including pestsfrom the Lygus genus. RNAi transgenes are provided for targeting thevacuolar ATPase H subunit, useful for controlling a coleopteran pestpopulation and infestation as described in US Patent ApplicationPublication 2012/0198586. PCT Publication WO 2012/055982 describesribonucleic acid (RNA or double stranded RNA) that inhibits or downregulates the expression of a target gene that encodes: an insectribosomal protein such as the ribosomal protein L19, the ribosomalprotein L40 or the ribosomal protein S27A; an insect proteasome subunitsuch as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasomebeta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomerof the COPI vesicle, the γ-coatomer of the COPI vesicle, the β′-coatomerprotein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2A protein which is a putative transmembrane domain protein; an insectprotein belonging to the actin family such as Actin 5C; an insectubiquitin-5E protein; an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport; an insectcrinkled protein which is an unconventional myosin which is involved inmotor activity; an insect crooked neck protein which is involved in theregulation of nuclear alternative mRNA splicing; an insect vacuolarH+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-bindingprotein. PCT publication WO 2007/035650 describes ribonucleic acid (RNAor double stranded RNA) that inhibits or down regulates the expressionof a target gene that encodes Snf7. US Patent Application publication2011/0054007 describes polynucleotide silencing elements targetingRPS10. PCT publication WO 2016/205445 describes polynucleotide silencingelements that reduce fecundity, with target polynucleotides, includingNCLB, MAEL, BOULE, and VgR. US Patent Application publication2014/0275208 and US2015/0257389 describes polynucleotide silencingelements targeting RyanR and PAT3. PCT publications WO/2016/138106, WO2016/060911, WO 2016/060912, WO 2016/060913, and WO 2016/060914 describepolynucleotide silencing elements targeting COPI coatomer subunitnucleic acid molecules that confer resistance to Coleopteran andHemipteran pests. US Patent Application Publications 2012/029750, US20120297501, and 2012/0322660 describe interfering ribonucleic acids(RNA or double stranded RNA) that functions upon uptake by an insectpest species to down-regulate expression of a target gene in said insectpest, wherein the RNA comprises at least one silencing element whereinthe silencing element is a region of double-stranded RNA comprisingannealed complementary strands, one strand of which comprises orconsists of a sequence of nucleotides which is at least partiallycomplementary to a target nucleotide sequence within the target gene. USPatent Application Publication 2012/0164205 describe potential targetsfor interfering double stranded ribonucleic acids for inhibitinginvertebrate pests including: a Chd3 Homologous Sequence, a Beta-TubulinHomologous Sequence, a 40 kDa V-ATPase Homologous Sequence, a EF1αHomologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, aJuvenile Hormone Epoxide Hydrolase Homologous Sequence, a SwellingDependent Chloride Channel Protein Homologous Sequence, aGlucose-6-Phosphate 1-Dehydrogenase Protein Homologous Sequence, anAct42A Protein Homologous Sequence, a ADP-Ribosylation Factor 1Homologous Sequence, a Transcription Factor IIB Protein HomologousSequence, a Chitinase Homologous Sequences, a Ubiquitin ConjugatingEnzyme Homologous Sequence, a Glyceraldehyde-3-Phosphate DehydrogenaseHomologous Sequence, an Ubiquitin B Homologous Sequence, a JuvenileHormone Esterase Homolog, and an Alpha Tubuliln Homologous Sequence.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest, either simultaneously orsequentially, with an insecticidally-effective amount of a recombinantIPD126 polypeptide of the disclosure. In some embodiments methods areprovided for killing an insect pest, comprising contacting the insectpest with an insecticidally-effective amount of one or more of arecombinant pesticidal protein of SEQ ID NOS: 19-36, or a variant orinsecticidally active fragment thereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population, eithersimultaneously or sequentially, with an insecticidally-effective amountof one or more of a recombinant IPD126 polypeptide of the disclosure. Insome embodiments, methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of one or more of a recombinant IPD126polypeptide of SEQ ID NOS: 19-36, or a variant or insecticidally activefragment thereof. As used herein, “controlling a pest population” or“controls a pest” refers to any effect on a pest that results inlimiting the damage that the pest causes. Controlling a pest includes,but is not limited to, killing the pest, inhibiting development of thepest, altering fertility or growth of the pest in such a manner that thepest provides less damage to the plant, decreasing the number ofoffspring produced, producing less fit pests, producing pests moresusceptible to predator attack or deterring the pests from eating theplant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population, either simultaneously or sequentially, with aninsecticidally-effective amount of one or more of a recombinant IPD126polypeptide of the disclosure. In some embodiments, methods are providedfor controlling an insect pest population resistant to a pesticidalprotein, comprising contacting the insect pest population with aninsecticidally-effective amount of one or more of a recombinant IPD126polypeptide of SEQ ID NOS: 19-36, or a variant or insecticidally activefragment thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof at leastone recombinant polynucleotide encoding a IPD126 polypeptide of thedisclosure. In some embodiments methods are provided for protecting aplant from an insect pest, comprising expressing in the plant or cellthereof a recombinant polynucleotide encoding one or more IPD126polypeptides of SEQ ID NOS: 19-36, or variants or insecticidally activefragments thereof.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, in certaininstances insects have evolved that are resistant to B. thuringiensisδ-endotoxins expressed in transgenic plants. Such resistance, should itbecome widespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis δ-endotoxins.

One way of increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) with transgenic crops producing a single insecticidalprotein active against target pests. The United States EnvironmentalProtection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the IPD126 polypeptides of the disclosure are usefulas an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins or other transgenes (i.e., anRNAi trait) including but not limited to Bt toxins, Xenorhabdus sp. orPhotorhabdus sp. insecticidal proteins, other insecticidally activeproteins, and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIPD126 polypeptide insecticidal proteins to insects in the orderLepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprises the presentation of at least one of theIPD126 polypeptides of SEQ ID NOS: 19-36, or variants or insecticidallyactive fragments thereof, insecticidal to insects in the orderLepidoptera and/or Coleoptera.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of at least one of the IPD126polypeptides to the insect species in combination with a secondinsecticidal protein to the insect species having different modes ofaction.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1. Coleoptera Diet-Based Feeding Assays

Western corn rootworm (WCRW, Diabrotica virgifera virgifera) bioassayswere conducted using the cell lysates 10 microliter samples mixed withmolten low-melt diet (Southland Products Inc., Lake Village, Arkansas)in a 96 well format. WCRW neonates were placed into each well of a 96well plate. The assay was run four days at 25° C. and then was scoredfor insect mortality and stunting of insect growth. The scores werenoted as dead, severely stunted (little or no growth but alive), stunted(growth to second instar but not equivalent to controls) or no activity.

Example 2. Strain Isolation, Cultivation and Activity Bioassay

Plant samples were collected from multiple locations in Iowa. Plantsamples were broken into smaller pieces and submerged in PBS buffer.After 15 min at low rpm shaking, 100 ul of the wash was then serialdiluted out and plated on several different isolation agar media.Various bacterial strains were picked and cultured in liquid Trypticasesoy medium (Tryptone 17 g/L, enzymatic digest of soy meal 3 g/L,Dextrose 2.5 g/L, Sodium Chloride 5 g/L, K2HPO4 2.5 g/L) overnight at26° C. with shaking at 250 rpm. The total protein was extracted fromcell mass and used for insect bioassay. Some strains, including Pantoeaagglomerans strain PMC3671E3-1 (NRRL Deposit No. B-67697), Pantoeaagglomerans strain PMC3671E9-1 (NRRL Deposit No. B-67698), and Pantoeaagglomerans strain PMCJ4082D4-1 (NRRL Deposit No. B-67699) showed stronginsecticidal activity against WCRW. The insecticidal activity wasfurther confirmed with new cultures.

Example 3. Strain Sequencing and Species Identification

Genomic DNA from strain PMC3671E9-1, PMC3671E3-1, and PMCJ4082D4-1 wasextracted with a Sigma Bacterial Genomic DNA Extraction Kit (Cat#NA2110-KT, Sigma-Aldrich, PO Box 14508, St. Louis, MO 63178) accordingto the manufactures' instructions. The DNA concentration was determinedusing a NanoDrop Spectrophotometer (Thermo Scientific, 3411 SilversideRoad, Bancroft Building, Suite 100, Wilmington, DE 19810) and thegenomic DNA was diluted to 50 ng/ul with sterile water. The genomes weresequenced with Illumina and PacBio sequencers. The sequences wereassembled and annotated. The 16S rDNA (SEQ ID NO: 37, SEQ ID NO: 38, andSEQ ID NO: 39) was extracted from the genome sequence and blastedagainst NCBI database. The results indicated that PMC3671E9-1,PMC3671E3-1, and and PMCJ4082D4-1 are Pantoea agglomerans strains.

Example 4. Active Protein Identification and Confirmation

This insecticidal activity of these strains exhibited heat andproteinase sensitivity indicating proteinaceous nature. Insecticidalactivity against WCRW was observed from both cell culture supernatantand clear cell lysate from Pantoea agglomerans strain PMC3671E9-1 grownin Terrific Broth and cultured for 2 days at 26° C. with shaking at 250rpm. 80 ml of Cell supernatant of PMC3671E9-1 was subjected toTangential Flow Filtration in a 100 kDa MWCO Polyethersulfone (PES)membrane from Spectrum Labs equilibrated in 20 mM Tris-HCl buffer, pH8.0, 150 mM NaCl (buffer A). The cell supernatant was concentrated, andbuffer exchanged into buffer A and a volume of 8 ml was recovered. Thismaterial was loaded onto a Superdex 200 26/600 pg column (sizeexclusion, GE Healthcare) equilibrated in buffer A. Fractionscorresponding with insecticidal activity were pooled and bufferexchanged into 1M Ammonium Sulfate, 20 mM Tris-HCl, pH 8 (buffer B) andapplied to a Source 15 Phenyl 4.6/100 column (hydrophobic interaction,GE Healthcare) equilibrated in buffer B. Protein was eluted with alinear gradient from 1 M to 0 M ammonium sulfate. Fractions weredesalted and subjected for identification of insecticidal activity.SDS-PAGE analysis of fractions with WCRW activity showed a bandcorresponding with insecticidal activity after staining with SYPRO rubygel stain (Invitrogen). Mass spectrometry was used to identify a fourgene operon encoded by strain PMC3671E9-1. The proteins were designatedas IPD126Aa-1, IPD126Aa-2, IPD126Aa-3 and IPD126Aa-4.

The PMC3671E9-1 DNA fragment containing IPD126Aa-1, IPD126Aa-2, IPD126-3and IPD126Aa-4 were subcloned into E. coli. The total protein extractfrom the transformed E. coli cells showed strong activity against WCRW,confirming the insecticidal activity of these proteins.

Example 5. Identification of Homologous Sequences

Genome sequences analysis indicated homolog sequences in other strains.Pantoea agglomerans strain PMC3671E3-1 contains all four IPD126 genesand have second copy of the first three genes upstream. Another Pantoeaagglomerans strain, PMCJ4082D4-1 (NRRL Deposit No. B-67699), is alsoactive on WCRW and contains two operons, similar to PMC3671E3-1. Thesecorresponding homolog sequences show 70 to 100% amino acid sequenceidentities to those in PMC3671E9-1.

Example 6. Live Culture Assay and Results

WCRW bioassays were conducting using live cultures of 20 ul samples andmolten artificial diet in a 96 well format. A serial dilution of theovernight PMC3671E3-1 culture was tested on multiple insect targets. Theculture and washed pellet showed killing activity against WCRW.

TABLE 1 WCRW Bioactivity PMC3671E3-1 culture Dilution SBL ECB FAW VBCCEW WCRW 1 x 0 0 0 0 0 3 1/2 x 0 0.5 0 0 0 3 1/4 x 0 0 0 0.25 0 3 1/8 x0 0.5 0 0 0 3 1/16 x 0 0 0 0 0 3 1/32 x 0 0 0 0.75 0 2.5 1/64 x 0 0 0 00 2 1/128 x 0.75 0.8 0 0 0 0 PMC3671E3-1 spent media SBL ECB FAW VBC CEWWCRW 1 x 0 0.75 0 0 0 1.5 1/2 x 0 0.25 0 0 0 1.5 1/4 x 0 0.25 0 0.75 0 11/8 x 0.25 0 0 0 0 1 1/16 x 0 0 0 0 0 0 1/32 x 0 0.5 0 0 0 0 1/64 x 0.250 0 0 0 0 1/128 x 0 0 0 0.75 0 0 PMC3671E3-1 washed cell pellet SBL ECBFAW VBC CEW WCRW 1 x 0 0 0 0 0 3 1/2 x 0 0.25 0 0 0 3 1/4 x 0 0.25 0 0 03 1/8 x 0 0 0 0 0 3 1/16 x 0.25 0 0 0 0 3 1/32 x 0 0.75 0 0 0 2.5 1/64 x0.25 0 0 0 0 1.83 1/128 x 0 0 0 0 0 1 Scores: 3-killing; 2-severestunting; 1-stunting; 1-no activity.

What is claimed is:
 1. A recombinant polynucleotide encoding an IPD126polypeptide comprising an amino acid sequence having greater than 95%sequence identity compared to the amino acid sequence of SEQ ID NO: 19and further comprising a heterologous regulatory sequence.
 2. Therecombinant polynucleotide of claim 1, wherein the recombinantpolynucleotide comprises the polynucleotide of SEQ ID NO:
 1. 3. A DNAconstruct comprising the recombinant polynucleotide of claim
 1. 4. Atransgenic plant or plant cell comprising the DNA construct of claim 3.5. A method of controlling Lepidoptera and/or Coleoptera insectinfestation in a transgenic plant and providing insect resistancemanagement, comprising expressing in the plant the polynucleotide ofclaim 1.